Quantitative determination of nitroglycerin in human plasma by capillary gas chromatography negative ion chemical ionization mass spectrometry

A quantitative method for determination of nitroglycerin in human plasma was developed. Nitroglycerin and the internal standard (butane-1,2,4-triyl trinitrate) were extracted from plasma with pentane. The extracts were analysed by gas chromatography mass spectrometry using fused silica capillary columns and electron capture negative ion chemical ionization. The quantitation limit of the method was about 50 pg ml-1. Linear calibration curves were obtained in the range of 50-1600 pg ml-1. Precision at the level of 100 pg ml-1 was 4%.     

Quantitative analysis of 6-hydroxymelatonin in human urine by gas chromatography-negative chemical ionization mass spectrometry

A method for assay of urinary 6-hydroxymelatonin, the major metabolite of the pineal hormone melatonin is described. After addition of an internal standard of deuterated 6-hydroxymelatonin sulfate, human urine was hydrolyzed enzymatically and free 6-hydroxymelatonin extracted, reacted to form a stable t-butyldimethylsilylpentafluoropropionyl derivative which was separated on silica gel column chromatography, and quantified using electron capture negative-ion chemical ionization mass spectrometry. Intrassay variability over an 18-h period was 5.4% [53.8 ng/3 ml urine ± 2.94 (SD)] and interassay variability over a 2-week period was 2.1% [51.8 ng/3 ml urine ± 1.08 (SD)].     

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.     

Analysis of histamine and N tau-methylhistamine in plasma by gas chromatography-negative ion-chemical ionization mass spectrometry

Current methods of quantitation of histamine and its major metabolite N tau-methylhistamine are inaccurate and insensitive to the very low concentrations that exist in plasma samples. Therefore, an accurate and sensitive method for quantification in plasma has been developed using the stable isotope dilution assay with negative ion-chemical ionization mass spectrometry. For histamine, after the addition of [2H4]histamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH2C6F5)3-histamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 430/434. For N tau-methylhistamine, after the addition of N tau-[2H3]methylhistamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the heptafluorobutyryl derivative (C3F7CO2)2-N tau-methylhistamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 497/500. The precision of the histamine assay is 3.1% and the accuracy is 95.5 +/- 2.5% while the precision of the N tau-methylhistamine assay is 1.9% and the accuracy is 106.8 +/- 1.9%. The lower limits of sensitivity are 1 pg for histamine and 6 pg for N tau-methylhistamine injected on column. Using the assay in three normal human volunteers, plasma concentrations of histamine were 130, 92, and 85 pg/ml, and of N tau-methylhistamine were 229, 228, and 216 pg/ml. This assay provides a very sensitive and accurate method of quantitation of histamine and N tau-methylhistamine in plasma samples.     

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.     

Determination of organophosphorus pesticide residues in human tissues by capillary gas chromatography-negative chemical ionization mass spectrometry analysis

We describe an analytical method that allows the determination of organophosphorus pesticides (OPs) in different human tissues. It involves an extraction procedure with ethanol-ethyl acetate, followed by gel permeation chromatography clean-up step and analysis by capillary gas chromatography-negative chemical ionization mass spectrometry in the selected ion monitoring mode. The method was tested for 37 OPs and the recoveries obtained vary between 60 and 106% with standard deviations ranging between +/-2 and +/-10. These values are independent of the analyzed tissue. Peak area repeatability as RSD for some OPs was < or =4.8% while a good linear relationship in the range 1.0-500 pg microl(-1) with r(2)> or =0.9878 was obtained. The limit of detection for the 37 OPs falls between 0.01 and 0.09 ng g(-1) with an RSD< or =9.5%. The analytical set up in this paper has been used to analyze different samples of human tissues (liver, healthy kidney, cancer kidney and adipose tissue) of 24 patients. The number of the identified OPs in the tissue samples is different (max. 20) according to the sample while their concentration ranges between the limit of detection and 28.0 ng g(-1). The highest concentrations have been determined in liver samples without any pathology (0.4-28.0 ng g(-1)) while the lowest concentrations have been determined in healthy kidney samples (0.01-1.50 ng g(-1)). In the cancer kidney samples OP concentrations vary between 0.03 and 4.6 ng g(-1): these concentrations are more elevated than those determined in healthy kidney samples. The comparison between the concentration of OPs determined in the healthy part, when possible, and those determined in the cancer part of the same kidney sample are very interesting: in fact, in the latter the OP concentration is generally 1-2-times higher than that in the former, an index of lower enzymatic activity in the cancer tissue.     

Profiling of prostaglandin biosynthesis in biopsy fragments of human lung carcinomas and normal human lung by capillary gas chromatography-negative ion chemical ionization mass spectrometry

Methods for the profiling of prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) biosynthesis in tissue samples of clinical origin by capillary gas chromatography-negative ion chemical ionization mass spectrometry (CGC-NICIMS) are detailed. Aliquots (25 microliter 1) of incubates (1 ml volume) of human lung carcinoma and normal human lung tissue fragments (total protein content = 0.2 to 2.0 mg) were derivatized for vapor phase analysis in the presence of 0.75 to 1.60 ng of tetradeuterated analogs of PGE2, PGF2 alpha and 6KPGF1 alpha without prior extraction and/or chromatography. The derivatized analytes and internal standards were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The inter-sample and intra-sample coefficients of variation for the assay method were typically less than 12%. The analysis of biological samples was completed with less than 2.5% of each derivatized sample per injection. The samples were of adequate purity for the identification and quantitation of each of the eicosanoids. The methods described in this report are highly selective and highly sensitive with detection limits of 0.1 to 0.2 picograms per injection. The analytical procedures provide the basis for comparisons of the qualitative and quantitative profiles of prostaglandin biosynthesis and should be adaptable for use in a variety of biological and clinical studies.     

Determination of polychlorinated biphenyl congeners in human milk by gas chromatography-negative chemical ionization mass spectrometry after sample clean-up by solid-phase extraction

This paper describes a simple and efficient procedure for measuring 25 congeners of polychlorinated biphenyls in human milk. The limit of quantitation was 0.1 ng/ml for five less chlorinated congeners (PCB 70, 74, 87, 99,101), and 0.01 ng/ml for the remaining 20 congeners (PCB 77, 105, 118, 126, 128, 138, 151, 153, 156, 169, 170, 180, 183, 187, 191, 194, 205, 206, 208 and 209). Solid phase extraction technology was applied to extract the analytes from the matrix and to remove lipids. Three columns were used sequentially, and they were a Bond Elut C(18), a Sep-Pak Plus NH2 and a Bond Elut PCB cartridge. The instrumental method was gas chromatography-mass spectrometry with negative chemical ionization, and selected ion monitoring mode was used.     

Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d₅ used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.     

Quantitative determination of atractylenolide III in rat plasma by liquid chromatography electrospray ionization mass spectrometry

Atractylenolide III is a major active component in Atractylodes macrocephala. This paper describes a simple, rapid, specific and sensitive method for the quantification of atractylenolide III in rat plasma using a liquid-liquid extraction procedure followed by liquid chromatography mass spectrometric (LC-MS) analysis. A Kromasil 3.5 microm C(18) column (150 mm x 2.00 mm) was used as the analytical column. Linear detection responses were obtained for atractylenolide III concentration ranging from 5 to 500 ng L(-1). The precision and accuracy data, based on intra-day and inter-day variations over 5 days were within 10.29%. The lower limit of quantitation for atractylenolide III was 5 ng mL(-1), using 0.1 mL plasma for extraction and its recoveries were greater than 85% at the low, medium and high concentrations. The method has been successfully applied to a pharmacokinetic study in rats after an oral administration of atractylenolide III with a dose of 20.0 mg kg(-1). With the lower limits of quantification at 5 ng mL(-1) for atractylenolide III, this method was proved to be sensitive enough for the pharmacokinetics study of atractylenolide III.     

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography-mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17beta]-2H(3)-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C(18) with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 microg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 microg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.     

Measurement of ergotamine in human plasma by triple sector quadrupole mass spectrometry with negative ion chemical ionization

By use of negative ion chemical ionization and collision-activated decomposition in a triple quadrupole mass spectrometer a method has been developed for the quantification of ergotamine in human plasma at levels down to 2 pg ml-1.     

Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization

A method is presented for the analysis of peptides in plasma at picomole to femtomole levels. Peptides are isolated from plasma by solid-phase extraction, the peptide of interest is purified by reversed-phase high-performance liquid chromatography (HPLC) and selectively digested using immobilized trypsin or chymotrypsin to yield specific di- or tripeptides. These di- and tripeptides are esterified using heptafluorobutyric anhydride, alkylated with pentafluorobenzyl bromide, then quantified by gas chromatography-mass spectrometry with negative ion chemical ionization. This method has been evaluated for a model synthetic heptapeptide, using a deuterium labeled analog as an internal standard. The half-life of the heptapeptide in human plasma was found to be 2 min. Extraction efficiencies of a tritiated peptide of similar size to the heptapeptide, [³H]DSLET, from plasma using either C₁₈ or strong cation-exchange columns were 85±3 and 70±2%, respectively. Quantitation of fragments from the heptapeptide indicated that the analysis was linear from 1–50 ng of the heptapeptide per ml of plasma. This method was subsequently employed for pharmacokinetic studies of the biologically active peptide Met-enkephalin-Arg-Gly-Leu, where linearity was obtained from 50 to 1000 ng/ml in rat plasma. This method demonstrated negligible side reaction by-products due to autolysis, and has potential for extensive use given the wide availability of gas chromatography-mass spectrometry.     

Quantitative determination of mitiglinide in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A selective, rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantitative determination of mitiglinide in human plasma. With nateglinide as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.2 mL plasma. The separation was performed on an ACQUITY UPLCtrade mark BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with the mobile phase consisting of methanol and 10 mmol/L ammonium acetate (65:35, v/v) at a flow rate of 0.25 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 1.080-5400 ng/mL, with a lower limit of quantification of 1.080 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was from -3.5% to 7.3% at all QC levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of mitiglinide in 10 healthy volunteers following oral administration.     

Quantitative determination of rivastigmine and its major metabolite in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive, specific, accurate and reproducible LC-MS-MS method was developed and validated for the simultaneous determination of rivastigmine and its major metabolite NAP 226-90 in human plasma according to International Regulatory Requirements. After addition of their respective labelled internal standards, the compounds were extracted from plasma using methyl-tert.-butyl ether at basic pH with a simultaneous derivatization of NAP 226-90 with propionic anhydride, and backextracted into an acidic solution. After re-extraction the compounds were analyzed on a 3-micrometer Purospher Star RP-18 column interfaced with a MDS Sciex API 3000 triple quadrupole mass spectrometer. Positive atmospheric chemical ionization was employed as the ionization source. The analytes and their internal standards were detected by use of multiple reaction monitoring mode. Intra- and inter-day accuracy and precision were found suitable over the range of concentrations between 0.200 and 30.0 ng/ml. The LC-MS-MS method was crossvalidated with a previously developed in-house GC-MS method by the analysis of plasma samples obtained from patients after administration of Exelon((R)) capsules and showed excellent correlation between the methods.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Quantitative determination of endogenous sorbitol and fructose in human erythrocytes by atmospheric-pressure chemical ionization LC tandem mass spectrometry

Evaluation of different extraction methods for quantification of endogenous sorbitol and fructose in human red blood cells (RBCs) and matrix effects in ESI and APCI showed that protein-precipitation followed by mixed-mode solid-phase extraction was more effective extraction method and APCI more effective ionization method. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical RBC samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitiol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with a linear range of 50.0-10,000 ng/mL (correlation coefficient >0.999) for sorbitol-13C6 and 250-50000 ng/mL (correlation coefficient >0.999) for fructose-13C6 in human RBCs.     

Gas chromatography-negative ion chemical ionisation mass spectrometry using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl derivatives for the quantitative determination of methylphenidate in human plasma

A novel electrophoric derivatisation procedure using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride for the quantitative determination of methylphenidate in human plasma is described. The drug can be quantitatively measured down to 0.006 pg/mL plasma due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with extraction solvent (n-hexane) and reagent solution for 15 min, which, after concentration was measured by GC-NICI-MS. The method is rapid as extraction and derivatisation occur in one single step. A stable isotope labelled internal standard was used. Validation data are given to demonstrate the usefulness of the assay, including selectivity, linearity, accuracy and precision, autosampler stability, aliquot analysis, robustness, and prospective analytical batch size accuracy. The method has been successfully applied to pharmacokinetic profiling of the drug after oral administration.     

Quantitative determination of nitroglycerol in human plasma by gas chromatography negative ion mass spectrometry using 15N labelled nitroglycerol as internal standard

Negative ionization resulted in the simplification of a previously published method. The new method permits the determination of 0.25 nmol l-1 nitroglycerol in plasma with a coefficient of variation of 9.1%.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.