Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard. Comparison with radioimmunoassay

Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.     

Assessment of serum cholesterol by two methods: gas-liquid chromatography on a capillary column and chemical ionization-mass fragmentography with isotopic dilution of [3,4-13C] cholesterol as internal standard.

A gas-liquid chromatographic (GLC) method and an isotopic dilution-mass fragmentographic (ID-MF) procedure using the same capillary chromatographic separation are described for serum cholesterol assay. GLC included silylation and separation on a highly efficient glass capillary column which allowed the separation of cholesterol from cholestanol and the use of epicoprostanol as internal standard. The concentrations were calculated from the areas of the signals and digitalized by a reporting integrator. The reproducibility was 0.5% and the correlation with the ID-MF technique was 0.997. The ID-MF technique was characterized by the use of [3,4-13C] cholesterol as the labelled standard and a chemical ionization mode. The reproducibility was 0.8%.     

Determination of cortisol in human plasma by capillary gas chromatography-mass spectrometry using [2H5] cortisol as an internal standard

A capillary gas chromatographic-mass spectrometric method for the determination of cortisol in human plasma using cortisol M + 5 as an internal standard is described. For calculation of plasma cortisol, peak areas were measured by selected-ion monitoring of the characteristic fragment ions of the dimethoxime-tri (trimethylsilyl) derivatives of cortisol and cortisol M + 5 (m/z 605 and 610, respectively). The inter- and intra-assay coefficients of variation for plasma sample were 3.07 and 1.77%, respectively. The method needed no complex corrections for contributions and provides a sensitive and reliable technique with good accuracy, precision and reproducibility.     

The mass spectra of [17-13C]phyllocladene and [3-13C]methylenecholestane—III

The mass spectra of [17-¹³C]phyllocladene and [3-¹³C]methylenecholestane have been examined. It is shown that there are some rearrangements at 70 e V as in the case of [17-¹³C]kaurene. However, no extensive randomization is evident at the molecular ion level. The results are interesting because very little is known about ¹³C randomization in polycyclic aliphatic hydrocarbons. The percentage retention of label was calculated for each ion.     

Quantitative determination of fluvastatin in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry using [18O2]-fluvastatin as an internal standard

A sensitive and specific method for the quantitative determination of fluvastatin in human plasma is presented. The drug was isolated from plasma by extractive alkylation with pentafluorobenzyl bromide and further derivatized to the bis-trimethylsilyl derivative. [18O2]-Fluvastatin was prepared from unlabelled fluvastatin and used as an internal standard. Gas chromatography/mass spectrometry under negative ion chemical ionization conditions was used for quantitative measurement of the drug, using m/z 554.26 and 558.26 for target and internal standard, respectively. Calibration graphs were linear within a range of 2 and 512 ng mL(-1) plasma. Intra-day precision was 0.94% (2 ng mL(-1)), 2.53% (8.2 ng mL(-1)), 2.16% (81.9 ng mL(-1)) and 3.26% (409.6 ng mL(-1)); inter-day variability was found to be 1.64% (2 ng mL(-1)), 0.97% (8.2 ng mL(-1)), 1.97% (81.9 ng mL(-1)) and 2.01% (409.6 ng mL(-1)). Intra-day accuracy showed deviations of 0.6% (2 ng mL(-1)), 0.37% (8.2 ng mL(-1)), -1.52% (81.9 ng mL(-1)) and -1.67% (409.6 ng mL(-1)); inter-day accuracy was of -1.64% (2 ng mL(-1)), -1.13% (8.2 ng mL(-1)), -2.28% (81.9 ng mL(-1)) and -0.46% (409.6 ng mL(-1)). The stable isotope labelled standard was found to be stable under the analytical conditions.     

Quantitative determination of galantamine in human plasma by sensitive liquid chromatography-tandem mass spectrometry using loratadine as an internal standard

A simple, rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method is developed and validated for the quantitation of galantamine, an acetylcholinesterase inhibitor in human plasma, using a commercially available compound, loratadine, as the internal standard. Following liquid-liquid extraction, the analytes are separated using an isocratic mobile phase on a reverse-phase C18 column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective (M+H)+ ions, m/z 288 to 213 for galantamine and m/z 383 and 337 for the internal standard. The assay exhibit a linear dynamic range of 0.5-100 ng/mL for galantamine in human plasma. The lower limit of quantitation is 0.5 ng/mL, with a relative standard deviation of less than 8%. Acceptable precision and accuracy are obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample makes it possible to analyze more than 400 human plasma samples per day. The validated method is successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability, or bioequivalence studies.     

Application of biosynthetic 13C-enrichment using [1-13C]-, [2-13C]- and [1,2-13C2]-acetate as precursor for 13C NMR spectral assignment of higher plant metabolites. The assignments of some bryonolic acid derivatives

The ¹³C NMR spectra of some derivatives of bryonolic acid (1) (D:C-friedoolean-8-en-3β-ol-29-oic acid) were assigned by means of ¹³C-enrichment, lanthanide-induced shifts (LIS) and comparison of chemical shift data between derivatives. The ¹³C-enriched species of 1, i.e., 1a, 1b and 1c were biosynthesized by Luffa cylindrica (Cucurbitaceae) callus fed with [1-¹³C]-, [2-¹³C]- or [1,2-¹³C₂]-acetate, respectively. Methyl acetylbryonolates 2, 2a, 2b and 2c, methyl bryonolates 3, 3a, 3b and 3c, methyl bryononates 4 and 4a, diacetyl-3β,29-diols (3,29-diacetyl-D:C-friedoolean-8-en-β,29-diol) 5, 5a, 5b and 5c, and 3-acetyl-3β,29-diols 6, 6a and 6b were prepared from 1, 1a, 1b and 1c, and their ¹³C NMR spectra were recorded. The ¹³C concentration of the ¹³C-enriched species was high enough to exhibit the satellite peaks clearly, and the analysed data were very useful for this study. Thus, total assignments for 2, 3, 4, 5 and 6 were established. It was found that conversion of the methoxycarbonyl group at C-29 into an acetoxymethyl group caused complex changes in the chemical shifts of the C, D- and E-ring carbons and those of the methyl carbons linked to these rings.     

Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantitation of formate by solid-phase microextraction and gas chromatography--mass spectrometry utilizing a [13C]formate internal standard

A new method for the analysis of formic acid was developed using gas chromatography-electron impact ionization mass spectrometry in the selected ion monitoring mode and solid-phase microextraction. Using this method with [13C]formic acid as an internal standard, the peak area ratio of [12C]formic acid/[13C]formic acid was not affected by differing methanol or sulfuric acid concentrations during the esterification and fiber adsorbing step. In comparison, the peak area ratio of formic acid/acetonitrile as detected by conventional GC with flame ionization detection was greatly affected by methanol or sulfuric acid concentrations. The formic acid calibration curve of our method showed excellent linearity over the range 5 to 200 microM. The within- and between-run assay relative standard deviations for the formic acid concentration were all less than 1.70%.     

Determination of glycerol by gas chromatography using meso-erythritol as internal standard

A simple chemical method for the determination of the glycerol moiety of triacylglycerols in lipids is described. The internal standard meso-erythritol is added and the mixture saponified in 0.5 M sodium hydroxide in methanol under reflux for 10 min. After cooling, the mixture is acidified with 2 M hydrochloric acid and the fatty acids extracted with hexane. The aqueous phase is then passed over a mixed cation-anion exchange resin, the eluate concentrated on the rotary evaporator below 40 [degree]C, dried overnight in a desiccator and glycerol plus meso-erythritol converted into their respective trimethylsilyl ethers by treatment with chlorotrimethylsilane and hexamethyldisilazane in pyridine. The silyl ethers are separated by capillary gas chromatography yielding the glycerol content of the lipid. As phospholipids yield mainly (>95%) glycerophosphate under our conditions of hydrolysis, which is subsequently removed by the mixed ion-exchange resin, the glycerol content is a measure of the triacylglycerol content of the original sample. A suitable correction for the presence of phospholipids can be applied by determining the phosphorus content of the lipids. In the majority of cases encountered this correction was negligible. The method is suitable for all compounds releasing glycerol on saponification, but it is especially useful for determining small amounts of triacylglycerols in an excess of wax esters. Examples of triacylglycerol determinations in the lipids of a number of South Atlantic fish species are presented.     

Determination of captopril in human blood by gas chromatography-negative-ion chemical ionization mass spectrometry with [18O4]captopril as internal standard

A method for the quantitative measurement of captopril in human blood is described. Blood was immediately treated with N-ethylmaleimide to prevent oxidative degradation. The carboxyl moiety was derivatized to the pentafluorobenzyl ester, which shows excellent properties for negative-ion chemical ionization mass spectrometry. A stable isotope-labelled standard was prepared from the intact target molecule in quantitative yield by exchanging the oxygen atoms of the free carboxylic acid and the imide moiety against 18O. The detection limit under negative-ion chemical ionization conditions is ca. 100 times lower than under electron-impact or positive-ion chemical ionization conditions, therefore only very small amounts of the original sample have to be analysed. The method was applied to be quantitative determination of unchanged captopril in human plasma after oral administration of a 25-mg dose.     

Determination of chlorophyll in plant samples by liquid chromatography using zinc-phthalocyanine as an internal standard

Chlorophyll analysis at high precision and accuracy is limited by the lack of suitable, commercially available internal standards for HPLC analysis. Here, the commercially available dye zinc-phthalocyanine is presented as a new internal standard to quantify chlorophylls in vegetable foods and to detect chlorophyll degradation products. The technique was applied to chlorophyll analysis of a selection of vegetable foods. Pigments were extracted with N,N-dimethylformamide from the vegetables and purified by solid phase extraction. Chlorophyll a, a', b, b', corresponding pheophytins, and zinc-phthalocyanine were separated by HPLC using a C18 reverse-phase column and fluorescence detection.     

Biosynthesis of the antibiotic verrucarin E use of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]-acetates. Verrucarins and roridins, 37th communication [1]

The origin of the carbon skeleton of verrucarin E (1) from acetate as precursor is confirmed. Incorporation studies with [1,2-¹³C]-acetate have demonstrated that two acetoacetate units couple together as shown in pattern A (Scheme 2) and not as in B . Analysis of the deuterium distribution in both verrucarin E (1) isolated after the incorporation of [2-¹³C,2-²H₃]-acetate and in sodium acetate obtained after Kuhn-Roth oxidation of the metabolite demonstrated that C(7) is derived from the starter unit of one of the acetoacetate moieties. The deuterium exchange in verrucarin E (1) occurring during fermentation was investigated.     

Fluorinated coal as an internal standard for unit mass assignment

Fluorination of bituminous coal by elemental fluorine yields a solid product from which a solid distillate can be made by vacuum pyrolysis. Both fluorinated materials are easily made and are useful for mass spectrometric unit mass assignments. They have certain advantages over the commonly used perfluorokerosene, particularly for use in the high mass region.     

Analysis of epoxyeicosatrienoic acids by chiral liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry using [13C]-analog internal standards

The metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) is thought to be mediated primarily by the cytochromes P450 (P450s) from the 2 family (2C9, 2C19, 2D6, and 2J2). In contrast, P450s of the 4 family are primarily involved in omega oxidation of AA (4A11 and 4A22). The ability to determine enantioselective formation of the regioisomeric EETs is important in order to establish their potential biological activities and to asses which P450 isoforms are involved in their formation. It has been extremely difficult to analyze individual EET enantiomers in biological fluids because they are present in only trace amounts and they are extremely difficult to separate from each other. In addition, the deuterium-labeled internal standards that are commonly used for stable isotope dilution liquid chromatography/mass spectrometry (LC/MS) analyses have different LC retention times when compared with the corresponding protium forms. Therefore, quantification by LC/MS-based methodology can be compromised by differential suppression of ionization of the closely eluting isomers. We report the preparation of [(13)C(20)]-EET analog internal standards and the use of a validated high-sensitivity chiral LC/electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the trace analysis of endogenous EETs as their pentafluorobenzyl (PFB) ester derivatives. The assay was then used to show the exquisite enantioselectivity of P4502C19-, P4502D6-, P4501A1-, and P4501B1-mediated conversion of AA into EETs and to quantify the enantioselective formation of EETs produced by AA metabolism in a mouse epithelial hepatoma (Hepa) cell line.     

Quantification of fexofenadine in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry using mosapride as internal standard

A rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of fexofenadine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 1-500 ng/mL for fexofenadine in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5% for fexofenadine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Simultaneous quantification of atorvastatin and active metabolites in human plasma by liquid chromatography-tandem mass spectrometry using rosuvastatin as internal standard

A simple, sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of atorvastatin and its active metabolites ortho-hydroxyatorvastatin and para-hydroxyatorvastatin in human plasma using rosuvastatin as internal standard (IS). Following simple liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 559/440 for atorvastatin, m/z 575/466 for ortho-hydroxyatorvastatin, m/z 575/440 for para-hydroxyatorvastatin and m/z 482/258 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for atorvastatin and its two metabolites in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin and the IS from spiked plasma samples were 54.2 +/- 3.2, 50.1 +/- 3.8, 65.2 +/- 3.6 and 71.7 +/- 2.7%, respectively. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.