Assays

Assays - Carnitine

 

Endurance exercise training and L-carnitine supplementation stimulates gene expression in the blood- und muscle cells in young athletes and middle aged subjects

Author: A. Lohninger, A. Sendic, E. Litzlbauer, R. Hofbauer, H. Staniek, D. Blesky, C. Schwieglhofer, M. Eder, H. Bergmüller, H.J. Mascher, D.G. Mascher
Publisher: Chemical Monthly 2005, 136, 1425-1442

Endurance exercise training is known to increase fatty acid (FA) oxidation during exercise and to stimulate mRNA synthesis of mitochondrial carnitine acyltransferases in skeletal muscle. To test the hypothesis that long-term endurance training induces cellular adaptions in different tissues, we determined the relative mRNA abundances of these genes in skeletal muscle and in blood cells from young athletes and middle aged untrained persons.

The first trial examined 6 cross-country skiers, at the start of high volume/low intensity exercise training and 6 months later, when training at the same exercise intensity had elicited a significantly slower rate of lactate accumulation.

In the second trial of 24 middle aged untrained (12 placebo and 12 carnitine supplemented) probands the mRNA expression was determined at the beginning and after three months of a low intensity endurance training program. A 5-fold increase of the muscle form of carnitine palmitoyltransferase 1 (CPT1B), a 4-fold increase in carnitine acetyltransferase (CRAT), and a 6-fold increase in the mRNA content of the main carnitine carrier OCTN2 in the muscles of the athletes were determined by reverse transcription quantitative real time polymerase chain reaction (RTPCR). The corresponding values examined in white blood cells were 12 (CPT1B), 4-(CTP1A), and 5-fold (OCTN2). In WBC of middle aged untrained subjects, the mRNA content of the liver form of the carnitine palmitoyferase 1 (CPT1A) was stimulated 2-fold (placebo group) and 8-fold in the carnitine supplemented probands. The relative abundances of CPT1B mRNA were increased by a factor of 3 (placebo) and 5 (carnitine supplemented), respectively. The mRNA abundances of OCTN2 increased 5-fold (placebo) and 7-fold (carnitine supplemented).

While the plasma carnitine levels also remained low during the study period in athletes, normal levels were determined in untrained subjects and significantly increased levels were found after carnitine supplementation. A marked increase of acylcarnitine excretion resulting in a reduction of the portion of short-chain acylcarnitines, other than acylcarnitine, due to carnitine supplementation could be interpreted as a detoxifying function.

The results of the present study may offer the opportunity to use the blood cells as a target for differential gene expressions and as an indicator for responses of skeletal muscle to exercise and / or nutrient supplementation.

Assays - Acetylcysteine

Endogenous plasma N-Acetylcysteine and single dose oral bioavailability from two different formulations as determined by a new analytical method

Author: B. Gabard, H.J. Mascher
Publisher: Biopharm. Drug Disposit. 12 (1991) 343 - 353

A method to quantitate N-acetylcysteine in plasma using reductive cleavage with tributylphosphine and post-HPLC column derivatisation with o-phtalaldehyde is described. Using this method, endogenous average concentrations of 0.08 µM were measured in 10 volunteers participating in a crossover study to compare the bioavailability of two different formulations of N-acetylcysteine. The drug was detected in plasma for up to 12 h after administration of a single oral dose (200 mg); the Cmax values were up to 20 times the endogenous levels. The sensitivity and selectivity of the method should thus enable the behaviour of N-acetylcysteine after oral administration to be properly described and bioavailability studies to be performed.

Assays - Acyclovir1

Pharmacokinetics and Bioavailability of Different Formulations of Aciclovir

Author: H. Vergin, H.J. Mascher, R. Metz
Publisher: Drug Research, 45 (1995), 508 - 515

The pharmacokinetics and bioavailability of aciclovir (CAS 59277-89-3) were examined after administration of newly developed 200 mg and 400 mg tablets. Two studies, each with 24 subjects of either sex, were performed. In the three-way study I, two different tablets containing 200 mg of aciclovir (test and reference products) and a short infusion of 250 mg aciclovir were compared. In the two-way study II, the bioequivalence of a newly developed 400 mg aciclovir tablet was tested against a standard product.

 

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New, high-sensitivity high-performance liquid-chromatographic method for the determination of Acyclovir in human plasma, using fluorimetric detection

Author: H.J. Mascher, C. Kikuta, R. Metz, H. Vergin
Publisher: J. Chromatogr., 583 (1992), 122-127

A high-performance liquid chromatographic method for the determination of acyclovir in human plasma has been developed. It is the first published chromatographic method capable of determining acyclovir in plasma with sufficient sensitivity and for sufficiently long periods of time following oral administration of a standard dose of acyclovir during pharmacokinetic investigations. Following precipitation of the proteins with perchloric acid, the sample is chromatographed with a strongly acidic mobile phase on a reversed-phase column, and is then subjected to fluorometric detection (excitation 260 nm, emission 375 nm). The determination limit is 6 - 10 ng/ml human plasma. The calibration is linear in the range of 10 - 12400 ng/ml plasma, with the coefficients of variation less than 8 %. the absolute recovery rate is between 102 and 113 %. This method has already been used to analyse several thousand plasma samples.

Assays - Acyclovir2

Pharmacokinetics and Bioavailability of Different Formulations of Aciclovir

Author: H. Vergin, H.J. Mascher, R. Metz
Publisher: Drug Research, 45 (1995), 508 - 515

The pharmacokinetics and bioavailability of aciclovir (CAS 59277-89-3) were examined after administration of newly developed 200 mg and 400 mg tablets. Two studies, each with 24 subjects of either sex, were performed. In the three-way study I, two different tablets containing 200 mg of aciclovir (test and reference products) and a short infusion of 250 mg aciclovir were compared. In the two-way study II, the bioequivalence of a newly developed 400 mg aciclovir tablet was tested against a standard product. << Back

Assays - Acylcarnitines

Long-term stability of amino acids and acylcarnitines in dried blood spots used for neonatal screening by tandem mass spectrometry

Author: K.A. Strnadová, M. Holub, A. Mühl, G. Heinze, R. Ratschmann, H.J. Mascher, S. Stöckler-Ipsiroglu, F. Waldhauser, F. Votava, J. Lebl, O.A. Bodamer
Publisher: Clin Chem 2007, 53, 717-722


Background: Dried blood filter cards, collected for newborn screening, are often stored for long periods of time. They may be suitable for the retrospective diagnosis of inborn errors of metabolism, but no data are currently available on the long-term stability of amino acids and acylcarnitine species.

Methods: We analysed amino acids and acylcarnitines by tandem mass spectrometry in 660 anonymous, randomly selected filter cards from 1989 through 2004. We assessed long-term stability of metabolites by linear regression and estimated annual decrease of concentration for each metabolite.

Results: Concentrations of free carnitine increased by 7.6 % per year during the first 5 years of storage and decreased by 1.4 % per year thereafter. Alanine, arginine, leucine, methionine, and phenylalanine decreased by 6.5 %, 3.3 %, 3.1 %, 7.3 %, and 5.7 % per year, respectively. Acetylcarnitine, propionylcarnitine, citrulline, gylcine, and ornithine decreased by 18.5 %, 27.4 %, 8.1 %, 14.7 %, and 16.3 % per year during the first 5 years, respectively; thereafter the decline was more gradual. Tyrosine decreased by 1.7 % per year during the first 5 years and 7.9 % per year thereafter. We could not analyze medium- and long-chain acylcarnitine species because of low physiological concentrations.

Conclusion: Estimation of the annual decrease of metabolites may allow for the retrospective diagnosis of inborn errors of metabolism in filter cards that have been stored for long periods of time.

 

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Endurance exercise training and L-carnitine supplementation stimulates gene expression in the blood- und muscle cells in young athletes and middle aged subjects

Author: A. Lohninger, A. Sendic, E. Litzlbauer, R. Hofbauer, H. Staniek, D. Blesky, C. Schwieglhofer, M. Eder, H. Bergmüller, H.J. Mascher, D.G. Mascher
Publisher: Chemical Monthly 2005, 136, 1425-1442

Endurance exercise training is known to increase fatty acid (FA) oxidation during exercise and to stimulate mRNA synthesis of mitochondrial carnitine acyltransferases in skeletal muscle. To test the hypothesis that long-term endurance training induces cellular adaptions in different tissues, we determined the relative mRNA abundances of these genes in skeletal muscle and in blood cells from young athletes and middle aged untrained persons.

The first trial examined 6 cross-country skiers, at the start of high volume/low intensity exercise training and 6 months later, when training at the same exercise intensity had elicited a significantly slower rate of lactate accumulation.

In the second trial of 24 middle aged untrained (12 placebo and 12 carnitine supplemented) probands the mRNA expression was determined at the beginning and after three months of a low intensity endurance training program. A 5-fold increase of the muscle form of carnitine palmitoyltransferase 1 (CPT1B), a 4-fold increase in carnitine acetyltransferase (CRAT), and a 6-fold increase in the mRNA content of the main carnitine carrier OCTN2 in the muscles of the athletes were determined by reverse transcription quantitative real time polymerase chain reaction (RTPCR). The corresponding values examined in white blood cells were 12 (CPT1B), 4-(CTP1A), and 5-fold (OCTN2). In WBC of middle aged untrained subjects, the mRNA content of the liver form of the carnitine palmitoyferase 1 (CPT1A) was stimulated 2-fold (placebo group) and 8-fold in the carnitine supplemented probands. The relative abundances of CPT1B mRNA were increased by a factor of 3 (placebo) and 5 (carnitine supplemented), respectively. The mRNA abundances of OCTN2 increased 5-fold (placebo) and 7-fold (carnitine supplemented).

While the plasma carnitine levels also remained low during the study period in athletes, normal levels were determined in untrained subjects and significantly increased levels were found after carnitine supplementation. A marked increase of acylcarnitine excretion resulting in a reduction of the portion of short-chain acylcarnitines, other than acylcarnitine, due to carnitine supplementation could be interpreted as a detoxifying function.

The results of the present study may offer the opportunity to use the blood cells as a target for differential gene expressions and as an indicator for responses of skeletal muscle to exercise and / or nutrient supplementation.

Assays - Allopurinol and Oxypurinol

A new and quick method for determination of Allopurinol and Oxipurinol in serum

Author: V. Nitsche, H.J. Mascher
Publisher: Arzneim.-Forsch./Drug Res. 30 (1980), 1855-1857

A method is described for allopurinol and oxipurinol assay in human serum for concentrations reached after usual therapy. The HPLC-method is based on a quick preparation of the serum samples including protein precipitation, evaporation to dryness, dissolving of the residue in citric acid and injecting the final solution on to the HPLC column. Detection limits for allopurinol are 0.05 µg/ml serum and for oxipurinol 0.2 µg/ml serum.

Assays - Amanitin

Quantitative Bestimmung von Amanitin im Serum mit HPLC

Author: A. Donner, H.J. Mascher, K. Hruby
Publisher: J. Clin. Chem Clin. Biochem. 25 (1987) 606

Assays - Amiloride1

Bioverfügbarkeit von Amilorid-Hydrochlorothiazid Kombinationspräparaten

Author: V. Nitsche, H.J. Mascher, H. Schütz
Publisher: Therapiewoche 36 (1985), 56-60

Assays - Amiloride2

Bioverfügbarkeit von Amilorid-Hydrochlorothiazid Kombinationspräparaten

Author: V. Nitsche, H.J. Mascher, H. Schütz
Publisher: Therapiewoche 36 (1985), 56-60

Assays - Aminobutyric acid

Overestimation of Serum Concentrations of Aminobutyric Acid in Patients with hepatic Encephalopathy by the Aminobutyric Acid-Radioreceptor Assay.

Author: P. Ferensci, J. Ebner, C. Kikuta et al.
Publisher: Hepatology 8 (1988) 69-72.

Assays - Amoxicillin

Determination of Amoxicillin in human serum and plasma by high-performance liquid chromatography and on-line postcolumn derivatization

Author: H.J. Mascher, C. Kikuta
Publisher: J. Chromatogr. A, 812 (1998), 221-226

A highly selective and fast HPLC method was developed for the determination of amixicillin in human serum and plasma. After protein precipitation with perchloric acid, an aliquot of the supernatant was neutralized by mixing it with sodium acetate solution and injected into a C18 HPLC column. Detection was done by a fluorescence detector after on-line postcolumn derivatisation with fluorescamine. The practical limit of quantification was 0.1 µg/ml using 0.3 ml of plasma. Linearity was given in the tested range of 0.1 to 15 µg/ml plasma. Inter-day precision (relative standard deviation) over 7 days for 0.42 µg/ml was ± 7.27 %; for 4.54 µg/ml, ± 5.24 % and for 13.18 µg/ml, ± 5.25 %. Stability over 50 days in serum and plasma occurs at -70°C but not at -20°C (-25 to -35 % reduction). This method was used for thousands of human serum and plasma samples.

 

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Determination of Amoxicillin [Amoxycillin] in plasma by high-performance liquid chromatography with fluorescence detection after online oxidation

Author: H.J. Mascher, C. Kikuta
Publisher: H. Mascher und C. Kikuta, J. Chromatogr. 506 (1990) 417-421

A simplified high-performance liquid chromatographic method for the determination of amoxicillin in plasma is described. Specific and sensitive fluorescence detection was achieved by on-line post-column electrochemical oxidation, using an electrochemical detector. Owing to the high specificity of the detection system, deproteinized plasma samples could be injected directly without prior treatment. This method permits a very fast and reproducible determination of amoxicillin in plasma on a routine basis at levels, down to 50 ng/ml. The absolute detection limit is about 10 pg injected.

Assays - Amphetamine

Determination of Desmethylselegiline, Methamphetamine and Amphetamine - the main metabolites of Selegiline in plasma by HPLC after derivatization

Author: H.J. Mascher, B. Göd, C. Kikuta
Publisher: J. Liqu. Chrom and Rel. Technol. 20 (1997), 797-809

Determination of desmethylselegiline, methamphetamine and amphetamine in plasma requires a very sensitive and selective method. These substances are metabolites of the MAO-inhibitor selegiline. This is the first published HPLC method which is able to determine these three substances down to a limit of quantification of 0.2 ng/ml human plasma. After extraction as free bases into an organic solvent and reextraction as salts into an inorganic acid all three substances and an internal standard (phentermine) are derivatized for fluorescence detection after reversed phase chromatography. The recovery is better than 90 % for all substances. The linearity in the range tested (0.2 - 15 ng for each substance/ml) is very good indeed. The precision and accuracy is usually smaller than 5 %. Pharmacokinetic results are presented.

 

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Pharmacokinetics and bioequivalence of the main metabolites of Selegiline: Desmethylselegiline, Methamphetamine and Amphetamine after oral aministration of Selegiline

Author: H. J. Mascher, C. Kikuta, A. Millendorfer, H. Schiel and G. Ludwig
Publisher: Int. J. Clin. Pharmacol. Ther. 35 (1997), 9 - 13

A bioavailability study of 2 different selegiline preparations were conducted in 20 healthy volunteers to test the bioequivalence. Almost no bioavailability study of selegiline has been published. As plasma levels of selegiline are very low and the elimination half-life is very short being about 9 minutes, therefore a very sensitive and selective method for determining the 3 main metabolites desmethylseligiline (DMS), methamphetamine (MA) and amphetamine (A) was developed. After application of a single oral dose of 5 mg selegiline the Cmax values of DMS reached 5 - 6 ng/ml, of methamphetamine (MA) 6 - 7 ng/ml and of amphetamine (A) about 2 ng/ml. The AUC∞ values were with DMS about 11 ng/ml x h, with MA about 130 ng/ml x h, and with A about 50 ng/ml x h. The 90 % confidence interval was with logarithmic transformed AUC∞ values 92 - 107 % with DMS, 89 - 107 % with MA, and 64 - 104 % with A. The logarithmic transformed Cmax values showed a 90 % confidence interval of 92 - 127 % with DMS, 91 - 101 % with MA, and 90 - 103 % with A. All relevant pharmacokinetic parameters showed bioequivalence with all 3 metabolites (DMS, MA, and A)

Assays - AP301

Publication

Assays - Bacitracin1

Determination of Neomycin and Bacitracin in Human or Rabbit Serum by HPLC-MS/MS

Author: D.G. Mascher, C.P. Unger, H.J. Mascher
Publisher: J. Pharm. Biomed. Anal. 2007, 43, 691-700

The method for the simultaneous determination of neomycin and bacitracin in human or rabbit serum was developed by using ion pairing reversed phase chromatography and tandem mass spectrometry (MS/MS) detection with electrospray (ESI) in positive mode. Both substances elute under these conditions at the same time and also kanamycin as internal standard elutes almost at the same time. The sample preparation was simple—only using 0.1 mL serum by protein precipitation with acetonitrile. Neomycin and bacitracin were detected as two-fold charged ions as well as the internal standard. The calibration range of these quite difficult detectable substances was 0.2–50 µg/mL of serum. The method was validated for both human or rabbit serum. The inter batch precision of quality control samples in human serum for neomycin ranged from 4.46 % to 8.99 % and for bacitracin from 6.85 % to 11.17 %. The inter batch accuracy for neomycin ranged from 98.7 % to 100.7 % and for bacitracin from 99.2 % to 103.0 %. At lower limit of quantitation (LLOQ) level of 0.2 µg/mL inter batch precision in human serum for neomycin was 12.05 % and for bacitracin 11.91 %, whereas accuracies were 99.9 % for neomycin and 102.7 % for bacitracin. Bench top stability in human or rabbit serum was given over three freeze thaw cycles and 4 h at room temperature. The method can be considered to be specific and recoveries for sample preparation were high.

Assays - Bacitracin2

Determination of Neomycin and Bacitracin in Human or Rabbit Serum by HPLC-MS/MS

Author: D.G. Mascher, C.P. Unger, H.J. Mascher
Publisher: J. Pharm. Biomed. Anal. 2007, 43, 691-700

The method for the simultaneous determination of neomycin and bacitracin in human or rabbit serum was developed by using ion pairing reversed phase chromatography and tandem mass spectrometry (MS/MS) detection with electrospray (ESI) in positive mode. Both substances elute under these conditions at the same time and also kanamycin as internal standard elutes almost at the same time. The sample preparation was simple—only using 0.1 mL serum by protein precipitation with acetonitrile. Neomycin and bacitracin were detected as two-fold charged ions as well as the internal standard. The calibration range of these quite difficult detectable substances was 0.2–50 µg/mL of serum. The method was validated for both human or rabbit serum. The inter batch precision of quality control samples in human serum for neomycin ranged from 4.46 % to 8.99 % and for bacitracin from 6.85 % to 11.17 %. The inter batch accuracy for neomycin ranged from 98.7 % to 100.7 % and for bacitracin from 99.2 % to 103.0 %. At lower limit of quantitation (LLOQ) level of 0.2 µg/mL inter batch precision in human serum for neomycin was 12.05 % and for bacitracin 11.91 %, whereas accuracies were 99.9 % for neomycin and 102.7 % for bacitracin. Bench top stability in human or rabbit serum was given over three freeze thaw cycles and 4 h at room temperature. The method can be considered to be specific and recoveries for sample preparation were high.

Assays - Benzbromarone

Detection of Benzbromarone in serum after combined therapy with Allopurinol

Author: V. Nitsche, H.J. Mascher
Publisher: Arzneim.-Forsch./Drug Res. 31 (1981), 510-512

A rapid method is described for benzbromarone assay in human serum. Protein precipitation is followed by extraction of the active substance. After centrifugation the clear organic layer is evaporated to dryness, redissolved in methanol and injected on a HPLC-column. Detection limits for this method of assay are 0.1 µg benzbromarone/ ml serum. The blood level for therapeutic concentrations lies between 1 and 2.5 µg/ml. Some pharmacokinetic data were presented. The half-life of benzbromarone is 2.6 h.

Assays - Carnitine

 

Endurance exercise training and L-carnitine supplementation stimulates gene expression in the blood- und muscle cells in young athletes and middle aged subjects

Author: A. Lohninger, A. Sendic, E. Litzlbauer, R. Hofbauer, H. Staniek, D. Blesky, C. Schwieglhofer, M. Eder, H. Bergmüller, H.J. Mascher, D.G. Mascher
Publisher: Chemical Monthly 2005, 136, 1425-1442

Endurance exercise training is known to increase fatty acid (FA) oxidation during exercise and to stimulate mRNA synthesis of mitochondrial carnitine acyltransferases in skeletal muscle. To test the hypothesis that long-term endurance training induces cellular adaptions in different tissues, we determined the relative mRNA abundances of these genes in skeletal muscle and in blood cells from young athletes and middle aged untrained persons.

The first trial examined 6 cross-country skiers, at the start of high volume/low intensity exercise training and 6 months later, when training at the same exercise intensity had elicited a significantly slower rate of lactate accumulation.

In the second trial of 24 middle aged untrained (12 placebo and 12 carnitine supplemented) probands the mRNA expression was determined at the beginning and after three months of a low intensity endurance training program. A 5-fold increase of the muscle form of carnitine palmitoyltransferase 1 (CPT1B), a 4-fold increase in carnitine acetyltransferase (CRAT), and a 6-fold increase in the mRNA content of the main carnitine carrier OCTN2 in the muscles of the athletes were determined by reverse transcription quantitative real time polymerase chain reaction (RTPCR). The corresponding values examined in white blood cells were 12 (CPT1B), 4-(CTP1A), and 5-fold (OCTN2). In WBC of middle aged untrained subjects, the mRNA content of the liver form of the carnitine palmitoyferase 1 (CPT1A) was stimulated 2-fold (placebo group) and 8-fold in the carnitine supplemented probands. The relative abundances of CPT1B mRNA were increased by a factor of 3 (placebo) and 5 (carnitine supplemented), respectively. The mRNA abundances of OCTN2 increased 5-fold (placebo) and 7-fold (carnitine supplemented).

While the plasma carnitine levels also remained low during the study period in athletes, normal levels were determined in untrained subjects and significantly increased levels were found after carnitine supplementation. A marked increase of acylcarnitine excretion resulting in a reduction of the portion of short-chain acylcarnitines, other than acylcarnitine, due to carnitine supplementation could be interpreted as a detoxifying function.

The results of the present study may offer the opportunity to use the blood cells as a target for differential gene expressions and as an indicator for responses of skeletal muscle to exercise and / or nutrient supplementation.

Assays - Carvone1

Pharmacokinetics of Carvone and Menthol after Administration of an Enteric Coated Formulation Containing Peppermint Oil and Caraway Oil

Author: H.J. Mascher, C. Kikuta, H. Schiel
Publisher: Wien Med Wochenschr. 2002, 152, 432-436

Enteric coating of peppermint oil/caraway oil capsules avoids subjective discomfort to the patient caused by gastroesophageal reflux. In order to confirm bioequivalence of an enteric coated formulation containing peppermint oil and caraway oil (Enteroplant®) and an immediate release formulation of both oils, the pharmacokinetics of menthol and carvone after oral administration of the two formulations were studied in a randomized, two-period cross-over study in 16 healthy male volunteers. The subjects received 180 mg peppermint oil and 100 mg caraway oil, once as 2 enteric coated capsules of the fixed combination preparation Enteroplant® containing 90 mg peppermint oil and 50 mg caraway oil each (test) and once in the form of 5 capsules of an immediate release formulation (reference) containing 36 mg peppermint oil and 20 mg caraway oil each. The capsules were taken with 250 ml water after a 10 h fast. Both substances were determined in serum by GC/MS after extraction. The limit of quantification was 10 ng/ml for menthol and 0.5 ng/ml for carvone. The mean maximum serum levels for menthol were 1196 ng/ml after administration of the test medication and 1492 ng/ml after administration of the reference medication. The bioavailability with respect to the AUC was comparable after administration of test and reference preparation, the 90 % confidence interval was 97 to 105 %. As expected, there were considerable differences for Tmax. After application of the enteric coated form the maximum concentration was reached significantly later (3.0 h vs. 1.7 h) compared to the immediate release capsule. Corresponding data were also calculated for carvone. After application of the test medication the maxima of 14 ng/ml for both formulations were reached later (2.5 h vs. 1.3 h). The 90 % confidence interval of the AUC for carvone was 79 % to 119 %, and therefore slightly outside the acceptable range for bioequivalence of 80 to 125 %. However, this fact should not be relevant, in particular since the dosage of the enteric coated capsule lies at the upper limit of the model text and positive clinical studies, also on the therapeutic equivalence of the two formulations, are available.

Assays - Carvone2

Pharmacokinetics of Carvone and Menthol after Administration of an Enteric Coated Formulation Containing Peppermint Oil and Caraway Oil

Author: H.J. Mascher, C. Kikuta, H. Schiel
Publisher: Wien Med Wochenschr. 2002, 152, 432-436

Enteric coating of peppermint oil/caraway oil capsules avoids subjective discomfort to the patient caused by gastroesophageal reflux. In order to confirm bioequivalence of an enteric coated formulation containing peppermint oil and caraway oil (Enteroplant®) and an immediate release formulation of both oils, the pharmacokinetics of menthol and carvone after oral administration of the two formulations were studied in a randomized, two-period cross-over study in 16 healthy male volunteers. The subjects received 180 mg peppermint oil and 100 mg caraway oil, once as 2 enteric coated capsules of the fixed combination preparation Enteroplant® containing 90 mg peppermint oil and 50 mg caraway oil each (test) and once in the form of 5 capsules of an immediate release formulation (reference) containing 36 mg peppermint oil and 20 mg caraway oil each. The capsules were taken with 250 ml water after a 10 h fast. Both substances were determined in serum by GC/MS after extraction. The limit of quantification was 10 ng/ml for menthol and 0.5 ng/ml for carvone. The mean maximum serum levels for menthol were 1196 ng/ml after administration of the test medication and 1492 ng/ml after administration of the reference medication. The bioavailability with respect to the AUC was comparable after administration of test and reference preparation, the 90 % confidence interval was 97 to 105 %. As expected, there were considerable differences for Tmax. After application of the enteric coated form the maximum concentration was reached significantly later (3.0 h vs. 1.7 h) compared to the immediate release capsule. Corresponding data were also calculated for carvone. After application of the test medication the maxima of 14 ng/ml for both formulations were reached later (2.5 h vs. 1.3 h). The 90 % confidence interval of the AUC for carvone was 79 % to 119 %, and therefore slightly outside the acceptable range for bioequivalence of 80 to 125 %. However, this fact should not be relevant, in particular since the dosage of the enteric coated capsule lies at the upper limit of the model text and positive clinical studies, also on the therapeutic equivalence of the two formulations, are available.

Assays - Cimetidine

New rapid assay of Cimetidine in human plasma by reversed-phase high-performance liquid chromatography.

Author: V. Nitsche, H.J. Mascher
Publisher: J. Chromatogr. 273 (1983), 449-452

Cimetidine is a potent inhibitor of gastric acid secretion and is used worldwide with increasing frequency in the treatment of duodenal and stomach ulcers. Earlier dosage directions of 1000 mg of cimetidine per day have now been reduced to values of 400 mg twice daily. It is therefore important to note not only the clinically demonstrable effect, but also the therapeutically effective serum levels. So far, high-performance liquid chromatographic data have been published only for normal phase chromatography, three papers on reversed phase, and one paper using a weak polar phase. The aim of this present publication is the elaboration of a rapid assay for cimetidine, which provides not only an easy procedure and a short analysis time, but also a high recovery rate, making it the method of choice for the determination of cimetidine in biological material, where this is linked with pharmacokinetic examinations.

Assays - Cinnarizine

Rapid high-performance liquid chromatographic assay of Cinnarizine in human plasma

Author: V. Nitsche, H.J. Mascher
Publisher: J. Chromatogr. 227 (1982), 521-525

Cinnarizine belongs to the category of antihistamines, more exactly to the sub-category of H1 receptor blockers. It is used in the treatment of cerebral and peripheral circulatory disturbances. Clinical experience of the use of cinnarizine goes back over a period of more than ten years. There is, however, little documentary material relating to pharmacokinetic studies and only a few publications have appeared on methods for cinnarizine assay in biological fluids. The present study describes a method of cinnarizine assay in serum or plasma employing high-performance liquid chromatography. The analysis can be carried out rapidly, it produces accurate results and it is thus especially suitable for routine examinations such as are involved in pharmacokinetic studies.

Assays - Codeine

Determination of Codeine in human plasma by reversed-phase high-performance liquid chromatography

Author: V. Nitsche, H.J. Mascher
Publisher: J. Pharm. Sci. 73 (1984), 1556-1558

Plasma from volunteers dosed with codeine was cleaned-up on a column of C18, from which codeine was eluted with methanol - 0.1 M HCl (1:1). Following addition of diazepam as internal standard, codeine was separated by reversed-phase HPLC on a column (25 cm x 4 mm) of Polygosil C18 (7.5 µm) operated at 45°C with methanol - 0.1 M ammonium carbonate (7:3) as mobile phase (2 ml/min) and detection at 220 nm. Calibration graphs were linear in the range 10 to 300 µg/l of codeine. Analysis took 3.5 min per run. The limit of detection was ~ 3 µg/l for 0.8 ml of plasma and a 40 µl loop. For a 150 µl loop and larger vol. of plasma, the detection limit can be reduced to < 1 µg/l. No interference was observed from drugs frequently co-administered with codeine. Recovery from doped samples was 98.4 ± 6.6 % (n = 16). Results of bioavailability studies agree well with those obtained by other methods, and the method is suitable for pharmacokinetic and clinical detection of codeine in plasma.

Assays - Desmethylselegeline

Determination of Desmethylselegiline, Methamphetamine and Amphetamine - the main metabolites of Selegiline in plasma by HPLC after derivatization

Author: H.J. Mascher, B. Göd, C. Kikuta
Publisher: J. Liqu. Chrom and Rel. Technol. 20 (1997), 797-809

Determination of desmethylselegiline, methamphetamine and amphetamine in plasma requires a very sensitive and selective method. These substances are metabolites of the MAO-inhibitor selegiline. This is the first published HPLC method which is able to determine these three substances down to a limit of quantification of 0.2 ng/ml human plasma. After extraction as free bases into an organic solvent and reextraction as salts into an inorganic acid all three substances and an internal standard (phentermine) are derivatized for fluorescence detection after reversed phase chromatography. The recovery is better than 90 % for all substances. The linearity in the range tested (0.2 - 15 ng for each substance/ml) is very good indeed. The precision and accuracy is usually smaller than 5 %. Pharmacokinetic results are presented.

 

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Pharmacokinetics and bioequivalence of the main metabolites of Selegiline: Desmethylselegiline, Methamphetamine and Amphetamine after oral aministration of Selegiline

Author: H. J. Mascher, C. Kikuta, A. Millendorfer, H. Schiel and G. Ludwig
Publisher: Int. J. Clin. Pharmacol. Ther. 35 (1997), 9 - 13

A bioavailability study of 2 different selegiline preparations were conducted in 20 healthy volunteers to test the bioequivalence. Almost no bioavailability study of selegiline has been published. As plasma levels of selegiline are very low and the elimination half-life is very short being about 9 minutes, therefore a very sensitive and selective method for determining the 3 main metabolites desmethylseligiline (DMS), methamphetamine (MA) and amphetamine (A) was developed. After application of a single oral dose of 5 mg selegiline the Cmax values of DMS reached 5 - 6 ng/ml, of methamphetamine (MA) 6 - 7 ng/ml and of amphetamine (A) about 2 ng/ml. The AUC∞ values were with DMS about 11 ng/ml x h, with MA about 130 ng/ml x h, and with A about 50 ng/ml x h. The 90 % confidence interval was with logarithmic transformed AUC∞ values 92 - 107 % with DMS, 89 - 107 % with MA, and 64 - 104 % with A. The logarithmic transformed Cmax values showed a 90 % confidence interval of 92 - 127 % with DMS, 91 - 101 % with MA, and 90 - 103 % with A. All relevant pharmacokinetic parameters showed bioequivalence with all 3 metabolites (DMS, MA, and A)

 

Assays - Dextrorphan and 3-Hydroxymorphinan

High-performance liquid-chromatographic determination of Dextrorphan and 3-Hydroxymorphinan in human plasma based on a selective pre-column sample cleanup

Author: H.J. Mascher
Publisher: J. Chromatogr. 420 (1987) 217-222

Dextromethorphan in a widely used non-narcotic antitussive agent. Its major metabolites are dextrorphan and 3-hydroxymorphinan. Since both substances are found in human plasma almost exclusively as glucuronides and sulphates, enzymic hydrolysis is a necessary part of the determination process. Several methods have been described for the determination of dextrorphan in human plasma, and one of these has also been used for the determination of 3-hydroxymorphinan. The assays were based on gas chromatography with flame-ionization detection, high-performance liquid chromatography (HPLC) with fluorescence detection)m HPLC with electrochemical detection and fluorimetry. Some of these procedures are very time-consuming, involving extraction, evaporation and lenghty chromatography; moreover, some of them lack selectivity and sensitivity.
This paper describes a procedure for the determination of dextrorphan and 3-hydroxymorphinan in human plasma, based on pre-column clean-up following enzymic hydrolysis. The first step is a selective sample clean-up on-line on a pre-column, the second step is the separation on a reversed-phase column with fluorescence detection. This procedure is highly sensitive: the limit of detection for dextrorphan and 3-hydroxymorphinan is as low as 0.2 ng/ml plasma. Moreover, this procedure is rapid, because the time-consuming process of extraction and evaporation are eliminated. The total analysis time after enzymatic hydrolysis is ca. 5 minutes, which permits analysis of as many as 80 samples per day.

Assays - Diclofenac

Comparison of UV and tandem mass spectrometric detection for the high-performance liquid chromatographic determination of diclofenac in microdialysis samples

Author: B.X. Mayer, K. Namiranian, P. Dehghanyar, R. Stroh, H. Mascher, M. Müller
Publisher: J Pharm Biomed Anal. 2003, 33, 745-754

High-performance liquid chromatography (HPLC) was used to analyse microdialysis samples obtained in vivo from human subcutaneous adipose tissue after topical application of the nonsteroidal anti-inflammatory drug diclofenac. For the reliable determination of diclofenac two different detection principles were applied in two different laboratories. One HPLC method utilized UV-detection at 280 nm, the other one used selected reaction monitoring mass spectrometry (MS). The HPLC-UV and -MS methods offered low limits of quantification of 10 and 1 ng/ml and an accuracy between 94.0 - 126.7 and 89.3 - 110.9 %, respectively. However, a comparison showed that the HPLC-UV method failed to determine diclofenac in biological matrices, as both false negative and positive values were found. HPLC-MS is clearly superior to HPLC-UV due to a much more selective detection, increased sensitivity and shorter run times.

 

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Topical skin penetration of Diclofenac after single- and multiple-dose application

Author: P. Dehghanyar, M. Brunner, B.X. Mayer, K. Namiranian, H.J. Mascher, M. Müller
Publisher: Int. J. Clin. Pharm. Ther. 2004, 42, 353-359

Objective: Transdermal penetration of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to be highly variable. The present study was performed to gain insight into the transdermal penetration process of topically applied diclofenac and to test whether transdermal absorption leads to pharmacologically effective concentrations in dermal tissue layers beneath the application site.
Material and method: Six healthy male volunteers participated in this 2-way crossover study and were assigned to 2 treatment groups. In the first group, diclofenac was applied in a therapeutic dose of 60 mg/100cm² 3 times daily for 4 days with subsequent occlusion with a plastic foil for 4 hours to enhance transdermal drug absorption. After a 1-week wash-out, diclofenac was applied at a single-dose of 300 mg/100cm² without occlusion. Diclofenac in both groups was applied on a previously shaven area of the thigh. Transdermal penetration was assessed employing in vivo microdialysis.
Results: After multiple-dose administration mean diclofenac concentrations of 0.48 ± 0.35 ng/ml were observed in subcutaneous tissue ( mean ± SEM). The mean AUC-subcutis/plasma-ratio of 0.08 ± 0.02 indicates redistribution of diclofenac from the systemic circulation to the tissue. After single-dose treatment, mean tissue concentrations were 24.26 ± 46.43 ng/ml with a mean AUCsubcutis/plasma ratio of 60.85 ± 57.59, which suggests direct tissue penetration of diclofenac.
Conclusions: Transdermal penetration of diclofenac after multiple as well as after single application of the present formulation is highly variable. In addition to other factors influencing the transdermal penetration process, dose and mode of administration are important factors determining whether pharmacologically effective local tissue concentrations are attained.

 

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Diclofenac concentrations in defined tissue layers after topical administration

Author: M. Müller, H. Mascher, C. Kikuta, S. Schäfer, M. Brunner, G. Dorner, H.G. Eichler
Publisher: Clin Pharmacol Ther., 293 - 299, 1997

To date is is unclear whether therapeutic concentrations are attained in target tissues after topical administration of nonsteroidal anti-inflammatory drugs. Therefore this study in healthy volunteers was undertaken to measure diclofenac concentrations attained in defined tissue layers directly underlying the site of topical diclofenac application by in vivo microdialysis. Methods: In each experiment two microdialysis probes were inserted, one into a superficial (3.9 mm) and one into a deep (9.3 mm) tissue layer, in 20 healthy volunteers and calibrated in vivo. The distance between the surfaces of the skin and the tips of the microdialysis probes were measured by 7.5 MHz ultrasound. Diclofenac was administered topically as a single dose of approximately 300 mg/100cm². Concentration versus time profiles in tissue layers were monitored for 5 hours. Results: Concentration versus time profiles in tissue layers were obtained in 11 of 20 experiments. However, there was no correlation between area under the concentration curve (AUC) in a defined layer and the depth of probe insertion. In those experimets where concentration versus time profiles were obtained for bith probes mean AUC was 532 µg x min / ml for superficial layers, and 438 µg x min / ml for deep layers. Conclusion: We conclude that transdermal penetration of diclofenac, at least single doses, is not predictible and may strongly be influenced by individual skin properties.

 

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The pharmacokinetics of a new sustained-release form of Diclofenac sodium in humans

Author: H.J. Mascher
Publisher: Drug Design and Delivery 4 (1989), 303-311

In the present study, two sustained release diclofenac preparations were administered every 12 hours over 4 days to ten human volunteers. Diurnal profiles were recorded on the 1st and 4th days, from which pharmacokinetic parameters were calculated: particular attention was given to cumulation. One, a newly develeped sustained release formulation, hat a MRT of 5.5 hours, and showed surprisingly small variation coefficients [AUC ss (72-84 hrs) Cmax (72-84 hrs)] after 7 administered doses; accordingly, the maximum concentrations were within a very narrow time window [tmax ss (72-84 hrs) range: 1.5 - 2.5 hours after administration]. Due to the selected release profiles with this formulation, there was no danger of cumulation in spite of administration every 12 hours [AUC 0-12 hrs, mean value 1555 ng/ml x h; AUC ss 72-84 hrs, mean value 1750 ng/ml x h].

Assays - Dihydralazine

Method Development of Dihydralazine with HPLC-MS/MS - an old but tricky substance - in Human Plasma

Author: D.G. Mascher, W. Tscherwenka, H.J. Mascher
Publisher: J. Pharm. Biomed. Anal. 2007, 43, 631-645

An HPLC-MS/MS method was developed and validated for the determination of dihydralazine in human plasma. HPLC-MS/MS has not been used before in a published paper and provides better sensitivity and selectivity. Therefore a much easier sample preparation than published before is feasible (protein precipitation). As this substance is rather reactive and sensitive some specific care has to be taken hindering the conversion of the substance in whole blood and following human plasma after blood withdrawal. Hydrazines often are used for derivatization of aldehydes and ketones. With specific care (using 1,4-dithiothreitol (DTT) and cooling) dihydralazine can be preserved and analysed without decomposition or conversion in the tested range of 0.500–302 ng/mL of human plasma. The following inter-batch precision and accuracy of the Quality Control Samples resulted: QC-A (1.34 ng/mL plasma) with a precision of coefficient of variation (CV) 7.66 % and an accuracy of 103.2 %; QC-B (18.2 ng/mL 7.86 %, acc. 101.3 %); QC-C (258 ng/mL, 9.73 %, acc. 98.3 %). The inter-batch values of the LLOQ samples at 0.500 ng/mL were 7.17 % for CV and accuracy of 106.4 %. Mean recovery tested at the QC levels was found to be 103.8 %. Specificity in six different plasma samples was good (< 10 % of the area of the LLOQ). Stability in plasma was tested under different conditions and was sufficient.

Assays - 5-Fluorouracil

Treatment of Advanced Colorectal Canceer With Folinic Acid and 5-Fluorouracil in Combination with Cisplatinum

Author: P. Sagaster, R. Essl, G. Teich, E. Fritz, M. Wasilewski, H. Umek, E. Dünser, H. Mascher, M. Micksche
Publisher: Eur. J. Cancer, 30A (1994), 1250 - 1254

51 patients with metastatic colorectal cancer (stabe Dukes D) were treated with intravenous (i.v.) infusion on days 1, 3, 5, 8, and 16 with folinic acid (200 mg/m²) and 5-fluorouracil (600 mg/m²), and on days 1, 8, and 16 with cisplatinum (25 mg/m² i.v.); cycles were repeated every 4 weeks. All 51 patients were evaluable for toxicity and response criteria. 26 patients had objective responses (3 complete responses, 5.9 %; 23 partial responses, 45.1 %), relative risk 51 % (95 % confidence intervals 36.7 - 65.0 %). Response duration ranged from 4 to 28.0 months (median 16.8). Overall median survival of all patients was 14.7 months (range 3.0 - 33.0). Toxicity of WHO grade III, requiring dose reduction, occurred in 9 (18 %) patients. The regimen described here appears to be active, safe and well tolerated for treatment of patients with advanced colorectal cancer.

Assays - Folinic acid and 5-MTHF

Treatment of Advanced Colorectal Canceer With Folinic Acid and 5-Fluorouracil in Combination with Cisplatinum

Author: P. Sagaster, R. Essl, G. Teich, E. Fritz, M. Wasilewski, H. Umek, E. Dünser, H. Mascher, M. Micksche
Publisher: Eur. J. Cancer, 30A (1994), 1250 - 1254

51 patients with metastatic colorectal cancer (stabe Dukes D) were treated with intravenous (i.v.) infusion on days 1, 3, 5, 8, and 16 with folinic acid (200 mg/m²) and 5-fluorouracil (600 mg/m²), and on days 1, 8, and 16 with cisplatinum (25 mg/m² i.v.); cycles were repeated every 4 weeks. All 51 patients were evaluable for toxicity and response criteria. 26 patients had objective responses (3 complete responses, 5.9 %; 23 partial responses, 45.1 %), relative risk 51 % (95 % confidence intervals 36.7 - 65.0 %). Response duration ranged from 4 to 28.0 months (median 16.8). Overall median survival of all patients was 14.7 months (range 3.0 - 33.0). Toxicity of WHO grade III, requiring dose reduction, occurred in 9 (18 %) patients. The regimen described here appears to be active, safe and well tolerated for treatment of patients with advanced colorectal cancer.

Assays - Formoterol

Ultra-sensitive determination of Formoterol in human serum by high performance liquid chromatography and electrospray tandem mass spectrometry

Author: D.G. Mascher, K. Zech, R. Nave, K.M. Kubesch, H.J. Mascher
Publisher: J Chromatogr. B 2006, 830, 25-34

An analytical method was developed and validated to determine Formoterol in human serum in the range from 0.40 to 100.24 pg/ml by high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) due to the lack of efficient methods to determine very low levels of Formoterol in serum and plasma. Serum was diluted by water and mixed with the internal standard (d6-Formoterol). Formoterol and internal standard were extracted using a cation-exchange solid phase column (SCX-3). After eliminating endogenous serum constituents through washing steps with water and methanol, elution took place using methanol/ammonia. After evaporation of the elution liquid the residue was redissolved and analyzed by HPLC-MS/MS with electrospray ionisation (ESI) in positive mode. A gradient between 10 mM ammonium formate and acetonitrile was used. The inter-batch precision of the calibration standards range from 1.55 % to 9.01 %. The inter-batch accuracy of the calibration standards ranged from 93.37 % to 107.30 %. The lower limit of quantitation (LLOQ, 0.40 ng/ml) had a precision of 19.67 % and an accuracy of 96.8 %. Comparable results were obtained for quality control samples. Stability in human serum was given over three freeze/thaw cycles and 2 h at room temperature. Formoterol in human serum was stable for at least 6 months below -20°C. This method has been used widely for quantifying Formoterol after inhalation of 9 - 36 µg of the drug by volunteers. A cross validation with human plasma versus serum was performed after this method was successfully validated in human serum.

Assays - 3-HPMA

High-performance liquid chromatographic-tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

Author: D.G. Mascher, H.J. Mascher, G. Scherer, E.R. Schmid
Publisher: J. Chromatogr. B, 750 (2001), 163 - 169


A sensitive and specific HPLC-tandem MS (HPLC-MS/MS) method was developed for the determination of 3-Hydroxypropylmercapturic acid in human urine. Samples were extracted using ENV+ cartridges and then injected on to a C8 Superspher Select B column with acetonitrile / 20 mM formic acid (1:19) as mobile phase. N-acetylcysteine was used as internal standard for HPLC-MS-MS. Linearity was obtained in the tested range of 50 - 5000 ng/ml of urine, with 50 ng/ml as the limit of quantification. Precision, as RSD, in the tested range of 50-5000 ng/ml was 1.47 - 6.04 %. Accuracy ranged from 87 to 114 %. 3-Hydroxypropylmercapturic acid was stable in human urine at 37°C for 24 h. The method was able to quantify 3-Hydroxypropylmercapturic acid in urine of non-smokers and smokers.

Assays - Ibuprofen enantiomers

Pharmacokinetics of Dexibuprofen administered as 200 mg and 400 mg film-coated tablets in healthy volunteers

Author: N. Eller, C. J. Kollenz, H. Schiel, C. Kikuta, H.J. Mascher
Publisher: Int. J. Clin. Pharmacol. Ther. 36 (1998), 414-417

The pharmacokinetic properties of 2 film-coated preparations containing 200 mg and 400 mg dexibuprofen were compared in a single-dose, crossover study in 16 healthy, male volunteers. Dexibuprofen was absorbed rapidly (tmax 2.1 - 2.2 hours) reaching the maximum concentrations of 12.4 µg/ml (200 mg), respectively 12.0 µg/ml (400 mg dose adjusted). For the characteristics AUC0-12 and AUC0-∞ arithmetic means of 49.2 µg x h / ml (200 mg) and 48.2 µg x h / ml (400 mg dose adjusted), respectively 50.5 µg x h / ml (200 mg), and 49.2 µg x h / ml (400 mg) were calculated. No relevant differences for the pharmacokinetic characteristics terminal half-life, clearance, volume of distribution, and mean residence time were observed. A linear dose-relationship was shown over the investigated dose range. Mean ratios after dosage adjustment of the test preparation using the “2 one-sided t-test” procedure were calculated. Bioequivalence was assessed for AUC0-12 with a mean ratio of 97.7 % (90 % CI: 92.4 - 103.3 %), for AUC0-∞ with 97.1 % (90 % CI: 91.4 - 103.1), and for Cmax with 97.5 % (90 % CI: 91.7 - 103.8 %). Both dexibuprofen preparations were well tolerated. No changes in haematological and biochemical parameters were detected.  

 

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Preliminary Toxicokinetic Study with Different Crystal Forms of S (+)-Ibuprofen (Dexibuprofen) and R,S-Ibuprofen in Rats

Author: S. Walser, R. Hruby, E. Hesse, H. Heinzl, H.J Mascher
Publisher: Arzneimittelforschung 47, 750 - 754, 1997

The aim of the study was to gain information on the plasma concentration-time profiles of both ibuprofen (CAS 15687-21-1) enantiomers in the rat after single oral application of two different crystal forms of S-(+)-ibuprofen (dexibuprofen, CAS 51146-56-6) and racemic ibuprofen in order to optimize blood-sampling times in a subsequent subchronic toxicity study. The application of either commercial racemic ibuprofen or recrystallised S-(+)-ibuprofen (60 mg/kg) to two groups of 4 rats per blood sampling term was carried out in order to define Cmax and tmax and AUC of the plasma concentrations of the ibuprofen enantiomers. The crystals of commercial (manufactured according to an usual manufacturing procedure) and recrystallised S-(+)- and racemic ibuprofen were different in respect to their shape and size. The recrystallised crystal species of S-(+)- and racemic ibuprofen has better galenic (tabletting-) properties and tablets containing the modified S-(+)-ibuprofen species showed favorable clinical results. The toxicokinetic behaviour of the recrystallised species was investigated in comparison to the commercial crystal species because of its slightly but significantly slower dissolution rat in simulated gastric and enteric juice. As the AUC0-24h of S-(+)-ibuprofen and the AUC0-24h of R-(-)-ibuprofen after application of commercial and recrystallised crystal species were not different, the crystal form apparently did not exert an influence on the extent of absorption of S-(+)-ibuprofen and racemic ibuprofen in the rat. The rat has a high inversion capacity and the inversion of R-(-)-ibuprofen after application of commercial and recrystallised racemic ibuprofen was nearly complete in this study. The effects of crystallinity on solubility in simulated media in vitro did not correlate to the findings on the extent of absorption in the rat in vivo.

 

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Comparison of the bioavailability of Dexibuprofen administered alone or as part of racemic Ibuprofen

Author: B. Gabard, G. Nirnberger, H. Schiel, H.J. Mascher, C. Kikuta, J.M. Mayer
Publisher: Eur. J. Clin. Pharmacol., 48 (1995), 505 - 511

Two bioavailability studies of S(+)-Ibuprofen (dexibuprofen) were conducted in healty volunteers to define the relationship between the bioavailibility of the drug after administration of dexibuprofen alone or as part of ibuprofen racemate. Enantionselective plasma drug analysis was used throughout. In the first study the bioavailability of dexibuprofen from a 400 mg tablet formulation was compared with that from 400 mg in aqueous solution. The tablet formulation did not influence the bioavailability of the drug and dexibuprofen was well absorbed from the gastro-intestinal tract. The second study was divided into three identical parts. Bioavailability of dexibuprofen 200, 400 and 600 mg was compared with its bioavailability from ibuprofen racemate 400, 800 and 1200 mg. The second study showed that the mean relative bioavailability of dexibuprofen to ibuprofen racemate was 0.66, thus enabling the estimation of clinically useful dexibuprofen doses from the usual doses of the racemate. The 95 % confidence interval limits did not include 0.5 leading to the conclusion that administering half of the racemate dose would not provide patients with an adequate amount of therapeutically active drug.

Assays - Indomethacin

Liquid-chromatographic determination of plasma Indomethacin in premature infants with patent ductus arteriosus

Author: A. Pollak, M. Weninger, H.R. Salzer, U. Salzermuhar, S. Levin, H.J. Mascher
Publisher: IRCS Medical science-biochemistry 14 (1986) 813-814

Indomethacin (I) and 8-methoxypsoralen (internal standard) were extracted from plasma with CHCl3 and separated by HPLC on a column of Microspher C-18 with a mobile phase of methanol - H3PO4 of pH 3.5 (3:1) and 320-nm detection. Response was linear from 0.7 to 4.0 mg/l and ≤75 µg/l of indomethacin could be detected. Recovery was 82% and inter-assay coefficient of variation were 6.8 and 3.9% for 150 and 385 µg/l of indomethacin, respectively.

Assays - Menthol

Pharmacokinetics of Carvone and Menthol after Administration of an Enteric Coated Formulation Containing Peppermint Oil and Caraway Oil

Author: H.J. Mascher, C. Kikuta, H. Schiel
Publisher: Wien Med Wochenschr. 2002, 152, 432-436

Enteric coating of peppermint oil/caraway oil capsules avoids subjective discomfort to the patient caused by gastroesophageal reflux. In order to confirm bioequivalence of an enteric coated formulation containing peppermint oil and caraway oil (Enteroplant®) and an immediate release formulation of both oils, the pharmacokinetics of menthol and carvone after oral administration of the two formulations were studied in a randomized, two-period cross-over study in 16 healthy male volunteers. The subjects received 180 mg peppermint oil and 100 mg caraway oil, once as 2 enteric coated capsules of the fixed combination preparation Enteroplant® containing 90 mg peppermint oil and 50 mg caraway oil each (test) and once in the form of 5 capsules of an immediate release formulation (reference) containing 36 mg peppermint oil and 20 mg caraway oil each. The capsules were taken with 250 ml water after a 10 h fast. Both substances were determined in serum by GC/MS after extraction. The limit of quantification was 10 ng/ml for menthol and 0.5 ng/ml for carvone. The mean maximum serum levels for menthol were 1196 ng/ml after administration of the test medication and 1492 ng/ml after administration of the reference medication. The bioavailability with respect to the AUC was comparable after administration of test and reference preparation, the 90 % confidence interval was 97 to 105 %. As expected, there were considerable differences for Tmax. After application of the enteric coated form the maximum concentration was reached significantly later (3.0 h vs. 1.7 h) compared to the immediate release capsule. Corresponding data were also calculated for carvone. After application of the test medication the maxima of 14 ng/ml for both formulations were reached later (2.5 h vs. 1.3 h). The 90 % confidence interval of the AUC for carvone was 79 % to 119 %, and therefore slightly outside the acceptable range for bioequivalence of 80 to 125 %. However, this fact should not be relevant, in particular since the dosage of the enteric coated capsule lies at the upper limit of the model text and positive clinical studies, also on the therapeutic equivalence of the two formulations, are available.

 

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Pharmacokinetics of Menthol and Carvone after Administration of an Enteric Coated Formulation Containing Peppermint Oil and Caraway Oil

Author: H.J. Mascher, C. Kikuta, H. Schiel
Publisher: Drug Res. (2001), 51 (I) 465-469

Enteric coating of peppermint oil/caraway oil capsules avoids subjective discomfort to the patient caused by gastroesophagael reflux. In order to confirm bioequivalence of an enteric coated formulation containing peppermint oil and caraway oil (CAS 277309-55-4, Enteroplant®) and an immediate release formulation of both oils, the pharmacokinetics of menthol and carvone after oral administrations of the two formulations were studied in a randomized, two-period cross-over study in 16 healthy male volunteers. The subjects received 180 mg peppermint oil and 100 mg caraway oil, once as 2 enteric coated capsules of the fixed enteric coated combination preparation containing 90 mg peppermint oil (WS® 1340) oil and 20 mg caraway oil (WS® 1520) each (test) and once in the form of 5 capsules of an immediate release formulation (reference) containing 36 mg peppermint (WS® 1340) oil and 20 mg caraway oil (WS® 1520) each. The capsules were taken with 250 mL water after a 10 h fast. Both substances were determined in plasma by GC/MS after extraction. The limit of quantification was 10 ng/ml for menthol and 0.5 ng/ml for carvone. The mean maximum plasma levels for menthol were 1196 ng/ml after administration of the test medication and 1492 ng/ml after administration of the reference medication. The bioavailability with respect to the AUC was comparable after administration of test and reference preparation, the 90 % confidence interval was 97 to 105 %. As expected, there were considerable differences for Tmax. After application of the enteric coated form the maximum concentration was reached significantly later (3.0 h bs. 1.7 h) compared to the immediate release capsule. Corresponding data were also calculated for carvone. After application of the test medication the maxima of 14 ng/ml for both formulations were reached later (2.5 h vs. 1.3 h). The 90 % confidence interval of the AUC for carvone was 79 to 119 % and therefore slightly outside the acceptable range for bioequivalence of 80 to 125 %. However, this fact should not be relevant, in particular since the dosage of the enteric coated capsule lies at the upper limit of the model text and positive clinical studies, also on the therapeutic equivalence of the two formulations, are available.

Assays - Methamphetamine

Determination of Desmethylselegiline, Methamphetamine and Amphetamine - the main metabolites of Selegiline in plasma by HPLC after derivatization

Author: H.J. Mascher, B. Göd, C. Kikuta
Publisher: J. Liqu. Chrom and Rel. Technol. 20 (1997), 797-809

Determination of desmethylselegiline, methamphetamine and amphetamine in plasma requires a very sensitive and selective method. These substances are metabolites of the MAO-inhibitor selegiline. This is the first published HPLC method which is able to determine these three substances down to a limit of quantification of 0.2 ng/ml human plasma. After extraction as free bases into an organic solvent and reextraction as salts into an inorganic acid all three substances and an internal standard (phentermine) are derivatized for fluorescence detection after reversed phase chromatography. The recovery is better than 90 % for all substances. The linearity in the range tested (0.2 - 15 ng for each substance/ml) is very good indeed. The precision and accuracy is usually smaller than 5 %. Pharmacokinetic results are presented.

 

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Pharmacokinetics and bioequivalence of the main metabolites of Selegiline: Desmethylselegiline, Methamphetamine and Amphetamine after oral aministration of Selegiline

Author: H. J. Mascher, C. Kikuta, A. Millendorfer, H. Schiel and G. Ludwig
Publisher: Int. J. Clin. Pharmacol. Ther. 35 (1997), 9 - 13

A bioavailability study of 2 different selegiline preparations were conducted in 20 healthy volunteers to test the bioequivalence. Almost no bioavailability study of selegiline has been published. As plasma levels of selegiline are very low and the elimination half-life is very short being about 9 minutes, therefore a very sensitive and selective method for determining the 3 main metabolites desmethylseligiline (DMS), methamphetamine (MA) and amphetamine (A) was developed. After application of a single oral dose of 5 mg selegiline the Cmax values of DMS reached 5 - 6 ng/ml, of methamphetamine (MA) 6 - 7 ng/ml and of amphetamine (A) about 2 ng/ml. The AUC∞ values were with DMS about 11 ng/ml x h, with MA about 130 ng/ml x h, and with A about 50 ng/ml x h. The 90 % confidence interval was with logarithmic transformed AUC∞ values 92 - 107 % with DMS, 89 - 107 % with MA, and 64 - 104 % with A. The logarithmic transformed Cmax values showed a 90 % confidence interval of 92 - 127 % with DMS, 91 - 101 % with MA, and 90 - 103 % with A. All relevant pharmacokinetic parameters showed bioequivalence with all 3 metabolites (DMS, MA, and A)

Assays - 5- and 8-Methoxypsoralen

5-Methoxypsoralen: Bioavailability and Pharmacokinetics

Author: V. Nitsche, H.J. Mascher
Publisher: Arzneim.-Forsch./Drug Res. 32 (1982), 1338-1341

The bioavailability of two galenic formulations of 5-methoxypsoralen (5-MOP) is described. A suspension in soft gelatine capsules was tested against a micronized powder in hard gelatine capsules on six volunteers, both in a dosage of 40 mg. The comparison of the AUC shows a significant better availability of the suspension (p ≤ 0.05). The detection limit of 5-MOP is 2 ng/ml plasma. The described analytical method is accurate and rapid - recovery near 100 % - and is suitable for routine analysis. The pharmacokinetic parameters for 5-MOP are 2.3 for the elimination half-life and 0.344 h-1 for the elimination rate constant.

Assays - Minocycline

Determination of Minocycline in human plasma by high-performance liquid chromatography with UV detection after liquid-liquid extraction

Author: H.J. Mascher
Publisher: J. Chromatogr. A, 812 (1998), 339 - 342

A sensitive and specific high-performance liquid chromatographic method was developed and validaten for the determination of minocycline in human plasma. The method uses liquid-liquid extraction, reextraction and HPLC with UV detection. The assay shows linearity in the tested range of 28 - 3533 ng/ml with a limit of quantification of 30 ng/ml. The inter-day precision was found to be ± 3.84 to ± 6.57 % (RSD) in the range of 148 to 2743 ng/ml. The method was successfully applied to pharmacokinetic studies.

Assays - Neomycin

Determination of Neomycin and Bacitracin in Human or Rabbit Serum by HPLC-MS/MS

Author: D.G. Mascher, C.P. Unger, H.J. Mascher
Publisher: J. Pharm. Biomed. Anal. 2007, 43, 691-700

The method for the simultaneous determination of neomycin and bacitracin in human or rabbit serum was developed by using ion pairing reversed phase chromatography and tandem mass spectrometry (MS/MS) detection with electrospray (ESI) in positive mode. Both substances elute under these conditions at the same time and also kanamycin as internal standard elutes almost at the same time. The sample preparation was simple—only using 0.1 mL serum by protein precipitation with acetonitrile. Neomycin and bacitracin were detected as two-fold charged ions as well as the internal standard. The calibration range of these quite difficult detectable substances was 0.2–50 µg/mL of serum. The method was validated for both human or rabbit serum. The inter batch precision of quality control samples in human serum for neomycin ranged from 4.46 % to 8.99 % and for bacitracin from 6.85 % to 11.17 %. The inter batch accuracy for neomycin ranged from 98.7 % to 100.7 % and for bacitracin from 99.2 % to 103.0 %. At lower limit of quantitation (LLOQ) level of 0.2 µg/mL inter batch precision in human serum for neomycin was 12.05 % and for bacitracin 11.91 %, whereas accuracies were 99.9 % for neomycin and 102.7 % for bacitracin. Bench top stability in human or rabbit serum was given over three freeze thaw cycles and 4 h at room temperature. The method can be considered to be specific and recoveries for sample preparation were high.

Assays - Nifedipine

HPLC determination of Nifedipine in plasma on normal phase

Author: H.J. Mascher und H. Vergin
Publisher: Chromatographia 25 (1988), 919-922

For the determination of nifepidine in plasma a sensitive and selective method is required. the use of on-line pre-column enrichment, followed by reversed phase separation and UV detection at 350 nm proved to be too susceptible. Therefore, detection was carried out after a post-column on-line reduction and photoreaction step using a fluorescence detector. Although this method proved to be extremely sensitive (limit of detection 0.1 ng/ml plasma) and selective, a number of problems cropped up caused by the reduction agent which finally prevented the procedure being used in routine analysis. As a consequence, the following method was developed. After liquid-liquid extraction of nifepidine 1/3 of the extract was chromatographed on a normal phase (diOH) system and detected at 235 nm, because detection at 350 nm was not sensitive enough. This method has a limit of detection of 1 ng nifepidine / ml plasma and the calibration curve is linear up to 320 ng/ml. The recovery lies around 82 % and the standard deviation for the range 6 - 320 ng/ml is less than 5 %. So far about 2000 plasma samples have been analysed by this method.

Assays - Norfloxacin

Determination of Norfloxacin in human plasma and urine by high-performance liquid chromatography and fluorescence detection

Author: H.J. Mascher, C. Kikuta
Publisher: J. Chromatogr. A, 812 (1998), 381-385

A method for the determination of norfloxacine in human plasma and urine is described. Plasma samples were deproteinized using acetonitrile. The supernatant was analysed by C18 HPLC. Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450 nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng/ml when 0.5 ml aliquots of plasma were handled. The intra-day precision of the spiked quality control samples ranged from ± 0.37 to ± 4.14 % in plasma (concentration range: 70.3 - 2109.2 ng/ml) and from ± 0.51 to ± 1.56 % in urine (concentration range 7.5 - 299.4 µg/ml). The intra-day accuracy obtained for norfloxacin in the quality control samples ranged from -5.18 % to -9.47 % in plasma and from -10.56 % to -5.91 % in urine. The assay has been used to support human pharmacokinetic studies.

Assays - Pantoprazole and metabolites

Bioavailability of a crushed Pantoprazole tablet after buffering with sodium hydrogencarbonate or magaldrate relative to the intact enteric coated Pantoprazole tablet

Author: L.M. Ley, B. Stahlheber-Dilg, P. Sander, R. Huber, H.J. Mascher, P.W. Lücker
Publisher: Methods Find Exp Clin Pharmacol. 2001, 23, 41-45

Assays - Paracetamol

Optimierte Bioverfügbarkeit von Paracetamol am Beispiel von ViCetamol-Brausepulver

Author: R. Riedlsperger, N. Eller, H. Schiel, H.J. Mascher
Publisher: Forum DR. MED 17/96

Assays - Peptide FX-06

The fibrin-derived peptide Bβ15-42 is cardioprotective in a pig model of myocardial ischemia-reperfusion injury

Author: J.P. Roesner, P. Petzelbauer, A. Koch, J. Mersmann, P.A. Zacharowski, O. Boehm, S. Reingruber, W. Pasteiner, D.G. Mascher, M. Wolzt, C. Barthuber, G.E.F. Nöldge-Schomburg, T.W.L. Scheeren, K. Zacharowski
Publisher: Crit Care Med 2007, 35, 1730 - 1735


Objective: The fibrin-derived peptide Bβ15-42 has been shown to reduce infarct size in rodent models of ischemia-reperfusion injury. To increase its potential for translation into the clinic, we studied the effects of Bβ15-42 in pigs, whose coronary anatomy is similar to that of humans. In addition, we evaluated the pharmacokinetics and safety of Bβ15-42 in several species, including humans.

Design: Animal study and phase I trial.
Setting: University hospital and contract research laboratories.
Subjects: Pigs / healthy volunteers.

Interventions: Male farm-bred Landrace pigs were subjected to 1 hr of left anterior descending coronary artery occlusion followed by 3 hrs of reperfusion. At the time of reperfusion, Bβ15-42 (2.4 mg/kg, n = 6) or random peptide (control; 2.4 mg/kg, n = 6) was administered as an intravenous bolus. As a positive control, pigs were subjected to ischemic preconditioning (n = 6). Cardiac damage and hemodynamics were recorded. Biodistribution and pharmacokinetics of Bβ15-42 were determined in rats and dogs. In a phase I trial involving 30 male healthy volunteers, pharmacokinetics and safety were tested in a randomized, double-blinded, placebo-controlled, parallel-group, single ascending dose study.

Measurement and Main Results: Bβ15-42 and ischemic preconditioning significantly reduced myocardial infarct size and troponin I levels. Bβ15-42 also reduces interleukin-6 levels, underlining its anti-inflammatory properties. Furthermore, in humans, the pharmacokinetics of the peptide Bβ15-42 were comparable to those of animals, and no serious adverse effects were observed.

Conclusions: Bβ15-42 elicits cardioprotection in pigs and is clinically safe in phase I testing of humans. This study confirms the new concept of a pathogenic role of fibrin derivatives in myocardial reperfusion injury, which can be inhibited by peptide Bβ15-42

Assays - Phenytoin

Comparative bioavailability of several Phenytoin preparations marketed in Austria

Author: V. Nitsche, H.J. Mascher, H. Schütz
Publisher: Int. J. Clin. Pharmacol., Ther. and Toxicol. 22 (1984), 104-107

In a multiple, randomized crossover study, plasma concentrations were determined following oral single-dose administration of various phenytoin preparations. With the exception of one formulations tested (p ≤ 0.01), no statistically significant difference was found among the remaining preparations.

Assays - Piracetam

Rapid method for the sensitive determination of Piracetam in plasma by high-performance liquid chromatography

Author: H.J. Mascher, C. Kikuta
Publisher: J. Pharm. Biomed. Analysis 7 (1989), 913-916

Piracetam (2-oxo-1-pyrrolidineacetamide) is a CNS-active substance, which was developed from GABA, an endogen active neurotransmitter substance. Piracetam influences interhemispherical exchange and protects the polyribosomal organism against oxygen-deficit states.
This paper describes a new and reliable HPLC method for the analysis of Piracetam, especially applicable to routine work. The analyses are performed by direct injection of plasma samples after deproteinization and employs UV-detection at 200 nm.

Assays - Pirenzepine

Bioavailability studies with Pirenzepine using a new HPLC-method

Author: H. Vergin, H.J. Mascher, K. Strobel und V. Nitsche
Publisher: Acta Pharmacologica et toxicologica 59 (1986), 207

 

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Pharmacokinetics and Bioequivalence of Different Formulations of Pirenzepine

Author: H. Vergin, H.J. Mascher, K. Strobel, V. Nitsche
Publisher: Arzneim.-Forsch./Drug Res. 36 (1986), 1409-1412
 

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Pharmacokinetics and Bioequivalence of Different Formulations of Pirenzepine

Author: H. Vergin, H.J. Mascher, K. Strobel, V. Nitsche
Publisher: Arzneim.-Forsch./Drug Res. 36 (1986), 1409-1412

An intraindividual comparative single-dose study was carried out under carefully controlled conditions on 12 healthy volunteers in order to establish the bioavailability of 5,11-dihydro-11-[(4-methyl-piperazin-1-yl)acetyl]-6H-pyrido[2,3b][1,4]benzodiazepin-6-one dihydrochloride (pirenzepine), the active principle of newly developed tablets (Gastricur ®) and suspension containing 10 mg. In an additional multiple dose, cross-over study on 12 healthy volunteers, the bioequivalence of pirenzepine was investigated after administration of the newly developed vs. commercial dose-equivalent tablets. Pirenzepine was assayed from plasma by a new, highly sensitive high-performance liquid chromatography method. A 3-compartment model was taken as a basis for the calculation of the plasma concentration curves and the pharmacokinetic parameters. Following i.v. administration, the terminal elimination half-life, the volume of distribution, and the total plasma clearance were determined to be 7.7 h, 0.255 l/kg and 263.4 ml/min, respectively. From the tablet and suspension formulation the systemic availabilities were calculated to be 33.5 % and 20. 3 %, respectively. In the multiple dose study, both tablet forms investigated were bioequivalent.

Assays - Pyridoxal

High-Performance Liquid Chromatography Determination of Total Pyridoxal in Human Plasma

Author: H. J. Mascher
Publisher: Methods Enzymol. Vol. 280 (Eds. D.B. McCormick, Academic Press 1997)

Vitamin B6 occurs in three interconvertible forms: pyridoxine (PN), pyridoxamine (PM), and pyridoxal (PL). There is also a 5´-phosphate for each of these forms (PNP, PMP, PLP). From the published method information on the endogenous levels of the vitamin analogs and their phosphated, it was apparent that PLP and PL might well constitute the majority of the vitamin analogs in plasma following administration of pyridoxine. We decided to determine PLP and PL as total PL following enzymatic cleavage with acid phosphatase. Preliminary trials had shown that sensitive analysis of PL was possible only with either extensive sample preparation or lang analysis times, because of the strong polarity of PL. For this reason we attempted to improve the selectivity and sensitivity of detection. Published papers prompted us to attempt postcolumn derivatization with semicarbazide.

 

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Determination of total Pyridoxal in human plasma following oral administration of vitamin B6 by high-performance liquid chromatography with post-column derivatization

Author: H.J. Mascher
Publisher: J. Pharm. Sci., 82 (1993), 972-974

An HPLC method for determining total pyridoxal from plasma was developed for a relative bioavailability comparison of two oral vitamin B6 (pyridoxine HCl) preparations. After cleavage of the pyridoxal-5-phosphate with the acid phosphatase enzym, the total pyridoxal was determined by HPLC. Pyridoxal was separated on a reversed-phase column, post-column derivatized to pyridoxal-semicarbazide, and then detected by fluorescence and quantified. The limit of detection was 2 ng/ml and interday variation (3 days) over the whole concentration range (13 - 215 ng/ml spiked) was < 4.1 %. In the relative bioavailability study, 16 human subjects were put on a low vitamin B6 diet for a period of 3 days. On the 2nd and 3rd days, 14 blood samples were taken per subject at the same times each day. The drug was administered on the 3rd day. Total endogenous pyridoxal detected on the 2nd day varied in plasma between 13 and 17 ng/ml. Pharmacokinetic parameters corrected with the Hoffmann-La roche preparation are, respectively: AUC0-24, 369.2 and 352.6 ng x h / ml; AUC24-48, 1638.2 and 1662.3 ng x h / ml; net Cmax, 193.0 and 197.1 ng/ml; tmax, 1.25 and 1.44 h; and relative bioavailability, 97.9 % (Westlake, 88 - 112 %)

 

Assays - Silibinin and Isosilibinin (with diastereomeric separation)

Diasteroisomeric separation of free and conjugated Silibinin in plasma by reversed-phase HPLC after specific extraction.

Author: H.J. Mascher, C. Kikuta, R. Weyhenmeyer
Publisher: J. Liqu. Chromatogr. 16 (1993), 2777-2789

The analytical method outlined herein makes it possible, for the first time ever, to detect the diastereomers of silibinin separately in human plasma following oral administration of silymarin or silibinin to human subjects in the course of pharmacokinetic investigations, with unprecedentedly low detection limits. The method permits detection of both free (= unconjugated) silibinin diastereomers and of silibinin diastereomers following enzymatic cleavage of the silibinin glucoronides and sulphates. The detection limit per diastereomer is 2.5 ng/ml for the free silibinin and 5 ng/ml following enzymatic cleavage. A crucial aspect of this method is its extremely selective extraction and re-extraction of the silibinin, with recovery rates of around 80 %. The diastereomers are separated, without derivatization, on a reversed phase C18-coluumn followed by UV detection at 285 nm. The linearity in the range tested (6 - 98 ng for each diastereomer / ml plasma in the case of free silibinin and 7 - 1829 ng for each diastereomer / ml plasma in the case of total silibinin) is very good indeed. The day to day variation (3 days, 3 concentrations; each n = 12) is lower than 4.8 % (CV) with an accuracy of -1.1 % to 6.1 %.

 

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Study on dose-linearity of the pharmacokinetics of Silibinin diastereomers using a new stereospecific assay

Author: R. Weyhenmeyer, H.J. Mascher, J. Birkmayer
Publisher: Int. J. Clin. Pharmacol., Ther. and Toxicol. 30 (1992), 134-138

Silibinin in single doses of 102, 153, 203 and 254 mg was applied as silymarin in capsules (Legalon ® 140) to 6 healthy male volunteers. Using a newly developed HPLC method, both diastereomers of silibinin were assayed in plasma as unconjugated compounds as well as total isomers after hydrolysis. In the dose range studies, the areas under the curves correlate linearly with the dose. On average, only 10 % of total silibinin in plasma is in the unconjugated form. The ratio of the silibinin isomers is reversed, if unconjugated and total isomers are compared. For unconjugated silibinin, the half-life is less than one hour, but the terminal half-life has probably not been observed, because already after 4-6 hours the levels fell below the limit of determination of 2.5 ng diastereomer/ml. For total silibinin, an elimination half-life of approximately 6 h is estimated. About 5 % of the dose is excreted into urine as total silibinin, corresponding to a renal clearance of approximately 30 ml/min. No adverse events were noted, showing that silymarin even in high doses, up to 5 capsules of Legalon ® 140, is well tolerated.

Assays - Tamoxifen

Specific high-performance liquid-chromatographic analysis of Tamoxifen and its major metabolites by "online" extraction and post-column photochemical reaction

Author: C. Kikuta, R. Schmid
Publisher: J. Pharm. Biomed. Analysis 7 (1989), 329-337

Plasma (0.8 ml) was mixed with 0.2 ml of 0.1M-HCl and then centrifuged for 2 min at 1200 g. For determination of tamoxifen (I) and demethyl-I, a portion (0.5 ml) of supernatant solution was applied to a pre-column (2.5 cm x 2 mm) of Sepralyte CN-propyl modified silica (40 µm) with H2O as mobile phase. The eluate was then applied to an analytical column (11 cm x 4.6 mm) of Partisil Si with a mobile phase of methanol - 5mM-ammonium acetate buffer (9:1). For the determination of 4-hydroxytamoxifen II, an analytical column (12.5 cm x 4 mm) of Lichrosorb RP-2 was used with a mobile phase of methanol - H2O - acetic acid - NN-dimethylhexamine (730:270:5:2). The separated compounds passed into a photoreactor (details given) and were converted into the corresponding phenanthrene by UV irradiation, and then detected fluorimetrically at 380 nm (excitation at 256 nm). The detection limits for I, demethyl-I and II were 100 pg/ml.

Assays - Thiamine

High-Performance Liquid Chromatography Determination of Total Thiamine in Human Plasma

Author: H. J. Mascher, C. Kikuta
Publisher: Methods Enzymol. Vol. 279 (Eds. D.B. McCormick, Academic Press 1997)

Thiamine lends itself to clinical and therapeutic use in the prevention and treatment of vitamin B1 deficiencies. Thiamine (T) is present endogenously, mainly in the form of various phosphates, of which thiamin diphosphate (pyrophosphate) predominates in plasma. For relative bioavailability studies, determination of total T is performed after separation of the different T phosphates by phosphatase. The literature contains only a few studies on the detection of T in rat or human blood or human plasma, especially determination in connection with bioavailability studies.

 

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High-Performance Liquid Chromatography Determination of Total Thiamine in human plasma for oral bioavailability studies

Author: H.J. Mascher, C. Kikuta
Publisher: J. Pharm. Sci., 82 (1993), 56-59

A high-performance liquid chromatographic method for the determination of total thiamine in plasma was developed for a relative bioavailability comparison of two oral thiamine preparations. After separation of thiamine mono- and diphosphates with the phosphatase enzyme, the total thiamine was subjected to reversed-phase high-peformance liquid chromatography, postcolumn oxidized to thiochrome with K3[Fe(CN)6], and detected by fluorescence. In the bioavailability study, 16 human subjects were put on a low-thiamine diet for 3 days. On the 2nd and 3rd days, 14 blood samples per subject and day were taken at the same times each day. Drug administration did not take place until the 3rd day. After deduction of the native concentrations of total thiamine detected on the 2nd day (mean value of ~ 7 ng/ml), the post-treatment pharmacokinetic parameters were determined (two different preparations, each with 200 mg of thiamine.HCl).

Assays - Triamterene and metabolite

Simple and fast HPLC method for the determination of Triamterene and Hydroxytriamterene sulfate in plasma and urine

Author: H.J. Mascher, M. Wasilewski
Publisher: J. Liqu. Chromatogr., 17 (1994), 1577-1585

Determination of triamterene (T) and hydroxytriamterenesulphate (HTS) in plasma and urine requires a very sensitive and selective method. As both of the substances are highly hydrophilic, it is hardly feasible to carry out an extraction. The method described obviates the need for pre-cleansing of samples. Plasma and urine samples, dilutet with water, are directly injected into the HPLC-column and analysed. For the detection of both of the substances fluorescence was used. The use of a Spherisorb-NH2-column with a mobile phase, consisting only of a buffer solution in water, makes it possible to dispense with protein precipitation of plasma. Both substances were analysed within 2 minutes in a single run. Detection limits of 1 ng/ml T and 20 ng/ml HTS in plasma as required in practice, were obtained without any difficulties. Referring to the precision of this method with plasma samples, the variation coefficient was below 3 % with HTS in the range of 20 to 1100 ng HTS / ml. Day to day variation showed with T in plasma values of smaller than 7 % in the range of 1 to 100 ng/ml.

Assays - Valproic acid

The Pharmacokinetics of Valproic Acid After Oral and Parenteral Administration in Healthy Volunteers

Author: V. Nitsche, H.J. Mascher
Publisher: Epilepsia 23 (1982), 153-162

The pharmacokinetics of valproic acid were investigated in six healthy volunteers. After a single intravenous dose of 1000 mg valproic acid, the pharmacokinetic parameters were determined according to the open two-compartment model. Bioavailability of valproic acid was performed comparing the areas und curves (AUC) after i.v. and an equal single oral dose. The half-life of the initial phase was 0.64 h, and the elimination half-life was calculated as 11.55 h. The distribution volume of the central compartment was 9.9 L, the apparent volume of distribution was 18.2 L, and the distribution volume at steady state was 12.6 L. The value for the average total clearance was 51.1 ml/min. The study showed that in comparison to single dosing, the elimination half-life increased in average for four hours after multiple dosing (p ≤ 0.05). There was only a poor correlation between the serum concentrations and concentration of valproic acid in saliva (r = 0.42).