Determination of acetyltrimoprostil and its metabolite trimoprostil in human or dog plasma by gas chromatography-negative-ion chemical ionization mass spectrometry

A sensitive and specific procedure is described for the determination of the antisecretory prostaglandin acetyltrimoprostil and its metabolite trimoprostil in human or dog plasma using gas chromatography--negative-ion chemical ionization mass spectrometry (GC--NICI-MS). Trideuterated analogues of both compounds are added to plasma as the internal standards. The plasma is extracted at pH 7.3 with benzene--dichloromethane (9:1), and the residue of the organic extract is reacted at room temperature with pentafluorobenzyl bromide in the presence of 18-crown-6-ether and potassium acetate. The derivatives are reconstituted in heptane, and appropriate aliquots are analyzed by GC--NICI-MS with selected-ion monitoring of the intense (M--C6F5CH2)- fragment ions of acetyltrimoprostil (m/z 419), trimoprostil (m/z 377), and their respective trideuterated analogues (m/z 422 and m/z 380, respectively). Quantitation of an experimental plasma sample is based on a comparison of the m/z 419 versus m/z 422 and m/z 377 versus m/z 380 ion ratios in each sample to that obtained from the analysis of drug-free plasma fortified with various amounts of both protio compounds, and a fixed amount of each trideuterated internal standard. The limit of quantitation of the assay for human plasma is 0.2 ng ml-1 with mean relative standard deviations at this concentration of 15.5% and 9.7% for acetyltrimoprostil and trimoprostil, respectively.     

Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry

A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.     

Trace level determination of phenols as pentafluorobenzyl derivatives by gas chromatography-negative-ion chemical ionization mass spectrometry.

A method for the trace level determination of 11 phenols as pentafluorobenzyl (PFB) derivatives by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical ionization (NICI) is described. First, the conditions for the PFB derivatisation of phenols were optimized and were found to be reaction temperature 80 degrees C and reaction time 5 h. Second, the detection limits using selected ion monitoring (SIM) were compared between trimethylsilylated (TMS) derivatives in the electron ionization (EI) mode and PFB derivatives in the NICI mode. The responses for the PFB derivatives in the NICI mode were 3.3-61 times higher than those of the TMS derivatives in the EI mode. The instrumental detection limits using NICI-SIM ranged from 2.6 to 290 fg. This method was applied to the analysis of phenols in river water using solid-phase extraction. The recoveries of the phenols from a river water sample spiked with standards at 100 ng l-1 with 2-chlorophenol, 4-chloro-3-methylphenol and pentachlorophenol and at 1000 ng l-1 with phenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 2-nitrophenol, 2,4,6-trichlorophenol and 4-nitrophenol were 81.2-106.3% (RSD 5.1-8.0%), except for 2-methyl-4,6-dinitrophenol and 2,4-dinitrophenol, for which the recoveries were 5.8 and 4.2%, respectively, because water contained in the acetone eluate interfered with the derivatisation of these compounds with two electrophilic nitro groups.     

Determination of clenbuterol in bovine plasma and tissues by gas chromatography-negative-ion chemical ionization mass spectrometry

A highly sensitive and specific assay was developed for the determination of clenbuterol in bovine plasma and tissues. Clenbuterol and the internal standard [2H9]clenbuterol were measured by gas chromatography-negative-ion chemical ionization mass spectrometry with methane as the reagent gas. Bovine tissues including muscle, liver, heart, kidney, lung, suet, brain, spinal cord and thymus were ground in a buffer of pH 7 and then extracted using ethyl acetate. After two subsequent purification steps, the cleaned-up organic extract was derivatized with pentafluoropropionic anhydride. The mass spectrometer was set to monitor the abundant ions m/z 368 and 377 of the perfluoroacyl derivatives. This assay was performed with 1 ml of plasma or 0.2 g of tissue. The feasibility of this method was demonstrated by the determination of clenbuterol residues as the femtomole level in a variety of tissues.     

Determination of calcium acetylhomotaurinate in human plasma and urine by combined gas chromatography-negative-ion chemical ionization mass spectrometry

A highly sensitive and specific assay has been developed for the determination of calcium acetylhomotaurinate and the internal standard (LM 3041) at the picomole level in human plasma and urine by gas chromatography-mass spectrometry with methane as the reagent gas. After a multiple-step extraction process, the cleaned-up organic extract was derivatized with pentafluorobenzoyl chloride at ambient temperature. Subsequently, chlorination followed by amidation of the sulphonic acid group led to the N-pentafluorobenzoyl di-n-butylamide derivatives. The mass spectrometer was set to monitor the abundant [M - HF]- ions (m/z 424 and 438), which were generated in the ion source switched in the negative-ion chemical ionization mode. This assay required 1 ml of plasma or 50 microliters of urine, and the detection limit was 1 ng/ml. The accuracy of the assay was tested day by day with quality control specimens spiked blind to the analyst. The mean difference between the theoretical and actual values was less than 8%.     

Quantification of 5-fluoro-2'-deoxyuridine in plasma by gas chromatography and negative-ion chemical ionization mass spectrometry.

A sensitive and specific method using gas chromatography and negative-ion chemical ionization mass spectrometry is described for the determination of 5-fluoro-2'-deoxyuridine (FdUrd) in plasma. The method is based on the formation of the pentafluoropropionyl derivative of FdUrd and of its stable isotope as internal standard after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography. Quantification in plasma was possible down to 300 pg/ml. The method was applied to the analysis of plasma levels of FdUrd in mice and dogs.     

Analysis of diethylstilbestrol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography-negative-ion chemical ionization mass spectrometry

A method has been developed for the detection of diethylstilbestrol, together with dienestrol and hexestrol, using extraction with a single immunoaffinity column containing antibodies raised against diethylstilbestrol, followed by gas chromatography-negative-ion chemical ionization mass spectrometry. Immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was synthesized by introducing a carboxyl group into the diethylstilbestrol molecule and coupling this product to bovine serum albumin. The columns were used for immunoadsorption of diethylstilbestrol and other estrogens, after dilution of samples with phosphate buffer, and were eluted with acetone-water (95:5 v/v). A derivatization method suitable for gas chromatographic-mass spectrometric analysis of diethylstilbestrol and other estrogens was developed using pentafluorobenzyl bromide and ethanolic potassium hydroxide as reagents. The derivatives obtained were detectable at the sub-picogram level using gas chromatography with negative-ion chemical ionization mass spectrometry. Recoveries of cis- and trans-diethylstilbestrol, dienestrol and hexestrol from the immunoaffinity columns, determined after extraction from urine, plasma and buffer, ranged from 28 to 96%. The sensitivity for diethylstilbestrol in urine samples was ca. 10 ppt. The method was applied to the analysis of urine from calves given a single dose of 10 mg of diethylstilbestrol. Free and glucuronic acid conjugated diethylstilbestrol decreased with time, but their ratio was variable.     

Determination of low plasma concentrations of 3-methoxy-4-hydroxyphenylethylene glycol using gas chromatography-negative-ion chemical ionization mass spectrometry

Gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICI-MS) allowed the detection of extremely low plasma concentrations of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG). Glucuronide and sulphate conjugates of MHPG were determined after enzymatic hydrolysis of plasma with beta-glucuronidase-arylsulphatase. A 1-ml plasma sample was extracted at the pH of the hydrolysis (pH 4.8) with ethyl acetate, and the dry extract was derivatized with pentafluoropropionic anhydride in ethyl acetate. After evaporation of the solvent, the residue was dissolved in benzene and an aliquot was analysed by GC-NICI-MS. A trideuterated analogue of MHPG was used as an internal standard. Negative-ion chemical ionization of the pentafluoropropionyl derivatives was carried out using ammonia. The ion-molecule adducts at m/e 766 and 785 (MHPG) and m/e 769 and 788 (internal standard) were formed from the pentafluoropropionyl derivatives with the ions of m/e 163 (CF3CF2COO-) and m/e 144 (loss of fluorine from m/e 163). The concentrations of the ions of m/e 163 and 144 play a major role in the sensitivity and precision of this technique, which allows the detection of free MHPG plasma concentrations as low as 100 pg/ml in routine analysis.     

Determination of estrogens in river water by gas chromatography-negative-ion chemical-ionization mass spectrometry.

A method for the determination of estrogens (17alpha-estradiol, 17beta-estradiol, estrone, ethynyl estradiol, and estriol) as pentafluorobenzyl-trimethylsilyl (PFB-TMS) derivatives by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical-ionization (NICI) is described. The NICI of all the derivatives produced an intense [M-PFB]- ion as the base peak. The reagent gas (methane) flow-rate and the ion source temperature were determined to be 2.0 ml/min and 240 degrees C, respectively, for the optimized NICI-selected ion monitoring (SIM) conditions. The sensitivities of the PFB-TMS derivatives in the NICI mode were 8.0-130 times higher than those of the PFB-TMS derivatives in electron ionization (EI) mode, and 12-25 times higher than those of all the TMS derivatives in the EI mode. This method was applied to the analysis of estrogens in river water using a solid-phase extraction as the sample preparation. The recoveries of the target chemicals from a river-water sample spiked with standards at 2 ng/l level were 85.8-126.5% (RSD, 6.2-13.0%). The methodical detection limits ranged from 0.10 to 0.28 ng/l.     

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.     

Simultaneous determination of prostanoids in plasma by gas chromatography-negative-ion chemical-ionization mass spectrometry

A method for simultaneous determination of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) in plasma was developed. After acidification and addition of 2H- and 3H-labelled internal standards, plasma prostanoids were extracted by reversed-phase cartridges and purified by normal-phase high-performance liquid chromatography. The pentafluorobenzyl, methoxime, trimethylsilyl derivatives were formed. Negative-ion chemical-ionization mass spectra with methane as reagent gas show one intense peak at m/z (M - pentafluorobenzyl). This ion was used for selective-ion monitoring. Prostanoid plasma concentrations (pg/ml) in five healthy volunteers were: PGE2 2.0-10.4, PGF2 alpha 2.2-9.8, 6-keto-PGF1 alpha 0.6-1.8, and TxB2 3.0-45.3. However, there is evidence that the TxB2 values may frequently be falsely high because of ex vivo production during the sampling procedure.     

Detection of phenolalkylamines by capillary column gas chromatography-negative chemical ionization mass spectrometry

Twelve phenolalkylamines normally used in different pharmaceutical products were determined in urine from volunteers given a therapeutical dose of the drug. The urine extracts were first derivatized with perfluoro propionic or butyric anhydride, then detected by capillary gas chromatography-negative chemical ionization mass spectrometry. The method is highly sensitive and specific in the analysis of biological samples.     

Detection of inorganic species by chemical reaction laser desorption ionization mass spectrometry

The oxidation numbers of metals in inorganic compounds were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by using their acetylacetonates, which are soluble in acetone. For the MALDI analysis of inorganic species insoluble in common solvents used for matrices, such as acetone, methanol, water, etc., a suspension method of sample preparation was developed. Turbid suspensions of inorganic species in the solvent were spotted on the sample holder with chelating reagents, as in the conventional sample preparation for MALDI-MS. Chemical reaction between the inorganic species and the chelating reagents occurred in the plume after irradiation by laser light. Metal oxides were also analyzed by this method, and samples with different oxidation numbers gave different mass spectra. These results suggest that many other metal oxides with different oxidation numbers could be identified if suitable chelating reagents are chosen for sample preparation for MALDI-MS.     

Quantification of acitretin in human plasma by microbore liquid chromatography-negative chemical ionization mass spectrometry

A highly sensitive liquid chromatographic-mass spectrometric procedure has been developed to quantitate plasma concentrations of acitretin, a dermatologic agent used to treat severe psoriasis. The assay utilizes the combination of normal-phase microbore high-performance liquid chromatography, negative chemical ionization mass spectrometry, selective ion monitoring and stable isotope dilution. The method has been used to measure acitretin and its metabolite, 13-cis-acitretin, over a range of 1-20 ng/ml in human plasma. The inter-assay precision was 5.3% for acitretin and 3.9% for 13-cis-acitretin, while the intra-assay precisions for acitretin and 13-cis-acitretin were 10.8 and 12.7%, respectively. Reproducibility of the assay for acitretin and 13-cis-acitretin, which was determined by the relative standard deviation of multiple analyses of the same quality assurance sample, was 5.9 and 8.1%, respectively.     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.