Simultaneous determination of benazepril hydrochloride and benazeprilat in plasma by high-performance liquid chromatography/electrospray-mass spectrometry

An analytical method for simultaneous determination of benazepril and its active metabolite, benazeprilat, in human plasma by high-performance liquid chromatography/electrospray-mass spectrometry was developed and validated. Rutaecarpine was selected as the internal standard. The separation was achieved on a C(18) column with acetonitrile and aqueous solution (0.1% formic acid) as mobile phase with a gradient mode. The quantification of target compounds was using a selective ionization recording at m/z 425.5 for benazepril, m/z 397.5 for benzeprilat and m/z 288.3 for rutaecarpine. The correlation coefficients of the calibration curves were better than 0.992 (n = 6), in the range of 6.67-666.67 ng/ml for benazepril and benazeprilat. The inter- and intra-day accuracy, precision, linear range had been investigated in detail. The method can be used to assess the bioavailability and pharmacokinetics of the drug.     

Determination of 17alpha-hydroxypregnenolone in human plasma by routine isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection

A first assay based on stable isotope dilution/gas chromatography-mass spectrometry has been developed for plasma 17alpha-hydroxypregnenolone, the leading marker of 3beta-hydroxysteroid dehydrogenase deficiency. Equilibration of plasma with internal standard was followed by solid phase extraction. After clean up using Sephadex LH-20 mini columns, heptafluorobutyrates were prepared as derivatives. Quantification was achieved by selected ion monitoring of m/z 467.0 (analyte) and m/z 471.0 (internal standard). 0.030 pmol of 17alpha-hydroxypregnenolone gave a signal to noise ratio of 3.7. Calibration plot was linear. Spiking experiments showed good accuracy with relative errors < 3.7%. Intraassay precision CV was 8.3% and interassay precision CV was 5.6%. Requiring small amounts of plasma, the rapid, convenient work up and the application of bench top GC/MS instrumentation proved our method suitable for routine clinical use in adults and children.     

Microdetermination of the 6,15-diketo-13,14-dihydroprostaglandin F1alpha in human plasma using gas chromatography/selected ion monitoring with [18O]6,15-diketo-13,14-dihydro-prostaglandin F1alpha as an internal standard

We devised a simple and effective purification method for the microdetermination of 6,15-diketo-13,14-dihydro-prostaglandin F1alpha (DK), a metabolite of prostacyclin (PGI2). [18O]DK was synthesized from the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O] water to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of DK. The methyl ester-methoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 613.4 for DK and at m/z 617.4 for an internal standard. A good linear response over the range of 10 pg to 10 ng was demonstrated. We detected DK to a level of about 297.8 pg/ml in human plasma. This method can be used to determine DK in biological samples.     

New diagnostic techniques for the detection of organic acidemias

We describe an improved procedure for the extraction of organic acids which is equally suitable for urine, plasma and amniotic fluid and gives similar high recoveries for a wide variety of organic acids, without interferences from phosphate, sulfate, or urea. Internal standards are added, the oxoacids are converted to the pentafluorobenzyloximes; the sample is concentrated, acidified, chromatographed on a small column of silicic acid, and the effluent neutralized and dried. After forming the trimethylsilyl derivatives, gas chromatography is done on a 0.53 mm X 30 m low-polarity bonded-phase fused silica capillary column. For gas chromatographic-mass spectrometric quantification, the peak areas at the m/z of a characteristic fragment ion for each acid relative to the peak areas of ions of the internal standards are related to standard curves.     

Determination of raltitrexed in human plasma by high performance liquid chromatography-electrospray ionization-mass spectrometry

A sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay was developed to determine raltitrexed in human plasma. After addition of benazeprilat as the internal standard (IS), methanol was used to produce a protein-free extract. Chromatographic separation was achieved with a Zorbax SB-C18 column (Narrow-Bore 2.1 mmx150 mm, 5-microm) using a mobile phase of acetonitrile-water containing 0.1% formic acid and 2% methanol (21.9:78.1, v/v). Raltitrexed was determined with electrospray ionization-mass spectrometry. HPLC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at [M+H]+ m/z 459.1 for raltitrexed and [M+H]+ m/z 397.1 for IS. Calibration curves were linear over the range of 2.0-3000 ng/ml. The lower limit of quantification was 2.0 ng/ml. The intra- and inter-batch variability values were less than 6.7% and 10.3%, respectively. The mean plasma extraction recovery of raltitrexed was in the range of 85.2-91.1%. The method was successfully applied to determine the plasma concentrations of raltitrexed in eight Chinese colorectal cancer patients.     

Liquid chromatographic method for the determination of uridine in human serum

A validated gas chromatography (GC)-mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)-tert-butyldimethylsilyl derivatives analyzed by GC-MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios (m/z) for the ions used for quantitation by single ion monitoring were 314 m/z for hydroxyproline, 198 m/z for pipecolic acid, 256 m/z for dehydroproline and 57 m/z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 micromol/l bone hydrolysate.     

Determination of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostan oic acid and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoi c acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry

11 alpha-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M) and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m/z 349 and m/z 356 (2H7 standard) (daughter ions of m/z 637 and m/z 644 (2H7 standard] were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m/z 484 and m/z 491 (2H7 standard) with the parent ions m/z 682 and m/z 689 (2H7 standard) were chosen.     

Validated method for quantitation of biomarkers for benzene and its alkylated analogues in urine

A validated gas chromatography-mass spectrometric method for the analysis of the metabolites of benzene and its alkylated analogues in urine is reported. A number of metabolites, as required by authorities for biomonitoring of industrial exposure to aromatic vapour, were analysed simultaneously with preservation of quantitative information concerning positional isomers. The use of this method replaces a combination of analytical methods required for the analysis of all these metabolites. Urine samples were subjected to acidic deconjugation followed by a derivatization step. Phenol, ortho-, meta-, para-cresol, mandelic acid, and ortho-, meta-, para-methylhippuric acid were analysed as their corresponding ethoxycarbonyl derivatives, with single ion monitoring. The mass-to-charge ratios (m/z) of the ions used for quantitation by single ion monitoring of the metabolites were: phenol, 94 m/z; cresols, 108 m/z; mandelic acid, 206 m/z; hippuric acid, 105 m/z; methylhippuric acids, 119 m/z. The mass-to-charge ratios for the internal standards were: [(2)H(6)]phenol, 99 m/z; p-chlorophenol, 128 m/z and 3-chloro-4-hydroxyphenyl acetic acid, 214 m/z. The limits of detection for phenol and the cresols were below 0.4 micromol/l and below 0.05 micromol/l for mandelic acid and the hippuric acids. Within-run precision for mandelic acid was 6.2%, for hippuric acid was 7.32% and was below 5% for the rest of the analytes.     

Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Simultaneous determination of imipramine and its metabolite desipramine in human plasma by capillary gas chromatography with mass-selective detection

An analytical method for the simultaneous determination of imipramine (IMI) and its N-desmethyl metabolite, desipramine (DIMI) in human plasma by capillary gas chromatography–mass selective detection (GC–MS), with D4-imipramine (D4-IMI) and D4-desipramine (D4-DIMI) as internal standards, was developed and validated. After addition of the internal standards, the compounds were extracted from plasma at basic pH into n-heptane–isoamyl alcohol (99:1, v/v), back-extracted into acidic aqueous solution and re-extracted at basic pH into toluene. Desipramine and D4-desipramine were converted into their pentafluoropropionyl derivatives. The compounds were determined by gas chromatography using a mass selective detector at m/z 234 for IMI, m/z 238 for D4-IMI, m/z 412 for DIMI and m/z 416 for D4-DIMI. The method was applied to clinical samples.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Quantitative analysis of albuterol in human plasma by combined gas chromatography chemical ionization mass spectrometry

A highly sensitive and specific quantitative assay for the determination of albuterol in human plasma, based on selected ion monitoring gas chromatography chemical ionization mass spectrometry, has been developed. The [MH]+ ions from the tri-TMS derivatives of albuterol (m/z 456) and the internal standard (2H3)albuterol (m/z 459), were assayed simultaneously by selected ion monitoring. The lower limit of quantitation is 0.25 ng ml-1 and the average assay precision (CV) for albuterol concentrations ranging from 0.25 ng ml-1 to 25 ng ml-1 is approximately 4%. This method is currently being employed for the routine quantitation of albuterol in plasma following the administration of doses therapeutically effective to man.     

Simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the positive/negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma. After addition of internal standards diazepam (for asperosaponin VI) and glycyrrhetic acid (for hederagenin), the plasma sample was deproteinized with acetonitrile, and separated on a reversed phase C18 column with a mobile phase of methanol (solvent A)-0.05% glacial acetic acid containing 10 mM ammonium acetate and 30 μM sodium acetate (solvent B) using gradient elution. The detection of target compounds was done in multiple reaction monitoring (MRM) mode using a tandem mass spectrometry equipped with positive/negative ion-switching ESI source. At the first segment, the MRM detection was operated in the positive ESI mode using the transitions of m/z 951.5 ([M+Na](+))→347.1 for asperosaponin VI and m/z 285.1 ([M+H](+))→193.1 for diazepam for 4 min, then switched to the negative ESI mode using the transitions of m/z 471.3 ([M-H](-))→471.3 for hederagenin and m/z 469.4 ([M-H](-))→425.4 for glycyrrhetic acid, respectively. The sodiated molecular ion [M+Na](+) at m/z 951.5 was selected as the precursor ion for asperosaponin VI, since it provided better sensitivity compared to the deprotonated and protonated molecular ions. Sodium acetate was added to the mobile phase to make sure that abundant amount of the sodiated molecular ion of asperosaponin VI could be produced, and more stable and intensive mass response of the product ion could be obtained. For the detection of hederagenin, since all of the mass responses of the fragment ions were very weak, the deprotonated molecular ion [M-H](-)m/z 471.3 was employed as both the precursor ion and the product ion. But the collision energy was still used for the MRM, in order to eliminate the influences induced by the interference substances from the rat plasma. The validated method was successfully applied to study the pharmacokinetics of asperosaponin VI and its active metabolite hederagenin in rat plasma after oral administration of asperosaponin VI at a dose of 90 mg/kg.     

In-source formation of N-acetyl-p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC-ECNICI-MS analysis

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M-PFB](-). In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen (N-acetyl-p-aminophenol, NAPAP-d(0); paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d(4); MW 155) in anhydrous acetonitrile and their GC-ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M-PFB](-) at m/z 150 from NAPAP-d(0) and m/z 154 from NAPAP-d(4), we observed highly reproducibly almost equally intense anions at m/z 149 and m/z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl-p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC-ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/z 149 to m/z 150 and of m/z 153 to m/z 154 decreased with increasing ion-source temperature in the range 100-250°C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI](-)) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC-EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC-MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.     

Analysis of tyrosine and deuterium labelled tyrosine in tissues and body fluids

A gas chromatographic mass spectrometric method has been developed to determine tyrosine and deuterium labelled tyrosine in biological samples using alpha-methyltyrosine (alpha-Me Ty) or m-hydroxyphenylalanine as internal standards. With the latter standard both labelled and unlabelled tyrosine as well as alpha-Me Ty can be determined simultaneously, in for example human blood, following administration of alpha-Me Ty to inhibit catecholamine synthesis. After isolation of the amino acids on an ion exchange column (Amberlite IR 120) the butyl ester pentafluoropropionyl derivatives were prepared. In human plasma the precision of the method was determined at a level of 80 nmol tyrosine ml-1 and found to be +/- 5% (coefficient of variation, n = 10).     

N-acetylglucosaminides. A new type of bile acid conjugate in man

Bile acids were extracted from human urine and were separated into groups of nonamidated and glycine- and taurine-conjugated compounds. Each group was subfractionated in a reversed-phase high performance liquid chromatography system, and the fractions were analyzed by negative ion fast atom bombardment mass spectrometry and also by gas chromatography-mass spectrometry after enzymatic removal of glycine and taurine moieties. The major glycosides of the non-amidated bile acids were more polar than reference bile acid glucosides and gave quasimolecular ions at m/z 592, 594, and 610 consistent with N-acetylglucosaminides of unsaturated dihydroxy and saturated di- and trihydroxy bile acids. Gas chromatography-mass spectrometry analyses of methyl ester trimethylsilyl ether derivatives showed fragments typical for N-acetylglucosaminides (m/z 173 and 186) in addition to those also given by glucosides (m/z 204 and 217). The N-acetylglucosaminides were inert toward alpha- and beta-glucosidase but were cleaved completely with N-acetylglucosaminidase. The released sugar moiety was identified as N-acetylglucosamine. One of the liberated bile acids was identified as ursodeoxycholic acid. The other acids were not identical to any known primary or secondary bile acid in humans. Fast atom bombardment mass spectrometry analyses of the glycine-and taurine-conjugated bile acid glycosides only showed ions consistent with the presence of glucosides (m/z 626 and 676). These compounds were sensitive only toward beta-glucosidase which liberated a trihydroxy bile acid as the major compound. Based on the recover of 13C- and 14C-labeled chenodeoxycholic acid glucoside added as internal standard, the daily excretion of nonamidated bile acid glycosides was estimated to be about 137 micrograms or 0.29 mumol, N-acetylglucosaminides constituting about 90%. The daily excretion of the glucosides of amidated bile acids was about 150 micrograms or 0.25 mumol, glycine conjugates constituting about 90%.     

Failure to verify the presence of pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase by gas chromatography/mass spectrometry

The presence of covalently bound pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase (BPAO) was examined by the use of gas chromatography/mass spectrometry. The enzyme was subjected to proteolysis with proteinase in the presence of [U-13C]PQQ as an internal standard. After isolation and derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ, respectively, by selected ion monitoring (SIM). In the SIM profile, although the sample extract obtained from BPAO treated with proteinase clearly showed the peak at m/z 462 for the internal standard, there were no peaks detectable at m/z 448, showing the absence of PQQ in the proteolysis digest of BPAO. Thus, our results do not support the claim that BPAO contains covalently bound PQQ in its structure.     

Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [²H₄]IAA (m/z 194, 134) or [²H₅]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [²H₃]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.