Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

An analytical method coupling online solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed to quantify 16 endogenous nucleoside mono- and triphosphates in cellular samples. Separation was achieved on a porous graphitic carbon (PGC) column without ion-pairing agent in the mobile phase. Low levels of the ion-pairing agent diethylamine (DEA) added to the reconstitution solution were necessary to prevent peak tailing of nucleoside triphosphates. The mass spectrometer, a triple quadrupole with an electrospray ionisation source, was operated in positive mode. Two multiple reaction monitoring (MRM) segments were programmed, each an internal standard. Extraction and separation of nucleoside mono- and triphosphates were obtained within 20 min. The total duration of a single run was 37 min. Calibration curves, performed with labelled nucleotides added to the sample matrix, ranged from 0.29 to 18.8 pmol injected for deoxyribonucleotides and from 3.9 to 3,156 pmol for ribonucleotides. Accuracy did not deviate more than ?14.6 and 10.2 % from nominal values for all compounds at all levels. CV results were all lower than 17.0 % for the LLOQ level and 14.6 % for the other levels. Quality control (QC) samples were also in agreement with acceptance criteria, except for the lower QC of GMP. Ion suppression, matrix effect, extraction recoveries and stability were assessed. After validation, the method was applied to the evaluation of the effects of gemcitabine and hydroxyurea on nucleotide pools in Messa cells.     

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.     

Quantitation of human glutathione S-transferases in complex matrices by liquid chromatography/tandem mass spectrometry with signature peptides

Direct quantitation of glutathione S-transferase (GST) isoforms [alpha (GST-A) and micro (GST-M)] in human liver cytosol was achieved by liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) analysis of signature peptides of GST-A and GST-M and their corresponding stable isotopic peptide internal standards via multiple reaction monitoring (MRM). The selection of signature peptides was performed via trypsin digestion of commercially available cDNA-expressed GST-A1 and GST-M1, followed by LC/ESI-MS/MS with an ion trap mass spectrometer and sequencing with the TurboSEQUEST application. Quantitative analysis of the selected signature peptides in the multi-reaction monitoring (MRM) mode was performed using a triple-quadruple mass spectrometer. A series of human cytosol samples was quantitatively analyzed for levels of GST-A and GST-M. The total level of GST-A and GST-M obtained from this LC/ESI-MS/MS method was well correlated with the total level of GST determined by the 1-chloro-2,4-dinitrobenzene (CDNB) method.     

Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry

The accurate and precise measurement of endogenous corticosteroids in urine is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate, and sensitive method based on liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the quantification of 67 endogenous gluco- and mineralo-corticosteroids and progestins has been developed and validated. Sample preparation, chromatographic separation, and mass spectrometric detection were optimized. Urine samples (0.5 mL) were hydrolyzed with β-glucuronidase and the released analytes were extracted by liquid–liquid extraction. The chromatographic separation was performed in 20 min after redisolution of the extract. MS behavior of endogenous corticosteroids was evaluated in order to select the most specific precursor ion ([M+H]⁺, [M+NH₄]⁺, or [M+H-nH₂O]⁺) for the detection. MS/MS determination was performed under selected reaction monitoring mode using electrospray ionization in positive mode. The method was shown to be linear (r > 0.99) in the range of endogenous concentrations for all studied metabolites. Limits of detection (LOD) below 1 ng mL⁻¹ were typically obtained for analytes with a 3-oxo-4-ene structure whereas LODs below 15 ng mL⁻¹ were common for the rest of analytes. Recoveries were higher than 80% and intra-assay precisions below 20%, evaluated at three concentration levels, were found for most steroids. No significant or moderate matrix effect, ranging from 54 to 155%, was observed for most of the analytes. The applicability of the method was confirmed by analyzing 24 h urine samples collected from twenty healthy volunteers and comparing the results with previously established normal ranges. The wide coverage of corticosteroid metabolism, together with short analysis time, low sample volume, simple sample preparation, and satisfactory quantitative results make this method useful for clinical purposes.     

Quantitation and validation of fluoroquinolones in eggs using liquid chromatography/tandem mass spectrometry

Fluoroquinolone antibiotics are labeled for very limited veterinary use for treatment of some animals and pets, but they are not authorized for food animals in Canada, including egg-producing birds. Because they are approved for other animals, however, fluoroquinolones may appear in eggs through off-labeling or accidental use. This method is capable of quantitating and confirming the presence of 4 fluoroquinolones, ciprofloxacin, danofloxacin, enrofloxacin, and sarafloxacin, over the concentration range 1 to 60 microg/kg (ppb) using 2 internal standards, norfloxacin or lomefloxacin. The compounds are extracted with acidic acetonitrile, isolated using Oasis HLB solid-phase extraction, and quantitated by liquid chromatography/tandem mass spectrometry at 2 transitions. The limits of detection (microg/kg) were evaluated from between-day experiments as: ciprofloxacin, 0.22; danofloxacin (m/z 314), 0.32; enrofloxacin, 0.22; and sarafloxacin, 0.31. The values for the decision limit, CCalpha were 0.33, 0.36, 0.30, and 0.45 microg/kg, respectively.     

Quantitation of simvastatin and its beta-hydroxy acid in human plasma by liquid-liquid cartridge extraction and liquid chromatography/tandem mass spectrometry

A sensitive and reliable procedure for the simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite (SVA) in human plasma was developed and validated. The analytes were extracted simultaneously from 0.5 ml aliquots of human plasma samples by methyl tert-butyl ether (MTBE) via Chem Elut cartridge extraction [also called liquid-solid extraction (LSE) or liquid-liquid cartridge extraction (LLCE)], separated through a Kromasil C(18) column (50 x 2 mm i.d. 5 microm) and detected by tandem mass spectrometry with a turbo ionspray interface. Stable isotope-labeled SV and SVA, (13)CD(3)-SV and (13)CD(3)-SVA, were used as internal standards. SV and SVA were detected in positive and negative ion modes, respectively, via within-run polarity switching. The use of Chem Elut cartridges not only provided a simple and efficient means of plasma sample extraction but also successfully reduced the interconversion between SV and SVA to an undetectable (for lactonization of SVA) or negligible (<0.07%, for hydrolysis of SV) level. The method showed excellent reproducibility, with intra- and inter-assay precisions <4.5% (RSD), and intra- and inter-assay accuracy between 94% and 107% of nominal values, for both analytes. The extraction recoveries were 78% and 87% on average for SV and SVA, respectively. The analyte was found to be stable in plasma through three freeze (-70 degrees C)-thaw (4 degrees C) cycles and for at least 3 h under bench-top storage condition in an ice-bath (4 degrees C), and also in the reconstitution solution at 4 degrees C for at least 24 h. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1) for both analytes, and has proved to be very reliable for the analysis of clinical samples.     

Quantitation of 5-fluorouracil (5-FU) in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

5-Fluorouracil (5-FU) has long had a place in the treatment of many malignancies. 5-FU plasma concentrations have been correlated with toxicity and efficacy, and therapeutic drug monitoring has been reported to result in an improved response/toxicity balance. We report validation, according to FDA guidelines, of a hydrophilic interaction chromatography (HILIC) liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of 5-FU in human plasma. The assay employed an isotopically labeled 5-FU internal standard and ethyl acetate extraction. Separation was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile/water (97:3, v/v), followed by a wash. Detection consisted of electrospray, negative-mode ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The accuracy was 96.0-102.2%, and precision was 2.1-7.5% in the concentration range of 10-10 000 ng/mL. Recovery from plasma was 46.0-72.6%, and ion suppression was 9.8-25.7%. Plasma freeze/thaw stability was 87.5-104.3%, and stability for 4 h at room temperature was 98.7-100.0%. This assay is currently being used to quantitate 5-FU in human plasma samples.     

Determination of miglitol in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the determination of miglitol in human plasma using voglibose as internal standard has been developed and validated. Samples of plasma were deproteinated with acetonitrile and washed with dichloromethane before being analyzed by reversed-phase high-performance liquid chromatography (HPLC). Separation was carried out on a short Nucleosil C(18) column (5 microm, 50 x 4.6 mm i.d.) using 10 mmol/L ammonium acetate at 1.0 mL/min as mobile phase. The detector was an Applied Biosystems Sciex API 4000 mass spectrometer using atmospheric pressure chemical ionization (APCI) for ion production. The instrument was operated at unit resolution in the multiple reaction monitoring mode. The assay was linear over the range 5.00-2000 ng/mL with a limit of detection of 1.00 ng/mL. Intra- and inter-day precision were <2.82% and <2.92%, respectively, with accuracy of 93.3-106%. The assay was successfully applied to a clinical pharmacokinetic study of miglitol given as a single oral dose (50 mg) to healthy volunteers.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

Quantitation of 8-hydroxydeoxyguanosine in DNA by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

A methodology has been developed and validated for quantifying 8-hydroxydeoxyguanosine (8-OHdG) in both commercial DNA and DNA isolated from livers of male Sprague-Dawley rats by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry. The analytical method conditions, including conditions for stabilizing 8-OHdG during complex nuclease P1 enzymatic digestion, were also evaluated. The limit of detection for 8-OHdG was 1.0 ng/mL (17.6 fmol on-column), and the linearity of the calibration curve was greater than 0.998 from 1.0 to 500 ng/mL. The intraday assay precision relative standard deviation (RSD) value for quality control (QC) samples was < or =5.59% with accuracies ranging from 91.84 to 117.61%. The interday assay precision (RSD) value was < or =1.76% with accuracies ranging from 91.84 to 116.67%. This method, combined with the LC/UV analysis of deoxyguanosine (dG), was used for determination of the levels of 8-OHdG/10(6) dG in DNA nuclease P1 enzymatic hydrolysates from both commercial DNA and rat liver DNA.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.     

Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry

An analytical method for the determination of total N-acetylcysteine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists of plasma digestion with dithiothreitol in order to reduce all the oxidized forms of N-acetylcysteine, and extraction with ethyl acetate followed by determination of levels by an LC–MS–MS method. The intra- and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 50 ng/ml of plasma. The concentration working range was established between 50 ng/ml and 1000 ng/ml. This method has been used in the analysis of approximately 800 human plasma samples from a clinical study with 24 volunteers; the precision of the quality controls was in the range 8.7 to 13.4% and the accuracy was in the range −5.9 to 8.5%, expressed as the RSD and the relative error, respectively.     

Quantification of tizanidine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (MS/MS) method was developed and validated for the assay of tizanidine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the selected reaction monitoring mode. The assay exhibited a linear dynamic range of 50-5000 pg/mL for tizanidine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 13%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. 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.     

Quantification of clopidogrel in human plasma by sensitive liquid chromatography/tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry method was developed and validated for the assay of clopidogrel in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 322/212 for clopidogrel and m/z 264/154 for the internal standard. The assay exhibited a linear dynamic range of 5-6000 pg/mL for clopidogrel in human plasma. The lower limit of quantification was 5 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 2.5 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.     

Determination of gatifloxacin in human plasma by liquid chromatography/electrospray tandem mass spectrometry

Gatifloxacin is an advanced-generation, 8-methoxyfluoroquinolone that is active against a broad spectrum of pathogens, including antiobiotic resistant Streptococcus pneumoniae. Development of a rapid, sensitive and selective method for the determination of gatifloxacin in human plasma is essential for understanding the pharmacokinetics of the drug when administered orally or intravenously. Solid phase extraction (SPE) using Oasis HLB was used to extract gatifloxacin and the internal standard ciprofloxacin from plasma. A method based on liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) was developed and validated to quantitate gatifloxacin in human plasma. The precursor and major product ions of the analyte were monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Mechanisms for the formation of collision-induced dissociation products of gatifloxacin are proposed. Linear calibration curves were generated from 10--1000 ng/mL with coefficients of determination greater than 0.99. The interday and intraday precision (%RSD) was less than 6.0% and accuracy (%error) was less than 5.4% for gatifloxacin. The limit of detection (LOD) for the method was 500 pg/mL based on a signal-to-noise ratio of 3.     

Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method

Quantitation of plasma angiotensin (Ang) II, the active mediator of the renin–angiotensin system, is challenging owing to its low physiological concentration. We report a validated liquid chromatography–mass spectrometry (LCMS) method to overcome this challenge. Ang II was extracted from EDTA plasma by an offline solid-phase extraction procedure with a Waters MAX μElution plate. LCMS quantitation was performed on the Waters TQS system, monitoring the 3+ ions of the peptide. The analytical performance of the LCMS method was validated. The stability of Ang II was studied with or without the presence of a protease inhibitor. Local reference intervals were established from 143 healthy normotensive subjects (57% female, 21–60 years old). The Ang II LCMS method had a measurable range of 3.3–700 pmol/L. The between-batch precision coefficient of variation was <7% over Ang II concentrations of 8.6–110 pmol/L. No significant matrix interference and carryover were observed. There was no significant difference in Ang II concentration in EDTA blood and plasma for at least 2h and 1 h at room temperature, respectively. Ang II was stable for at least 1 year when stored at −80°C, with or without the protease inhibitor. Age-dependent Ang II reference intervals were established: 4.4–17.7 pmol/L (21–30 years) and 3.9–12.8 pmol/L (31–60 years). The present LCMS method is suitable for quantitation of plasma Ang II to study the renin-angiotensin system.