A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-β glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.     

Development and validation of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of exemestane in human plasma

Exemestane, irreversible steroidal aromatase inhibitor, acts as a false substrate for aromatase enzyme and significantly lowers circulating estrogen concentrations in postmenopausal women with hormone-sensitive breast cancer. A sensitive bioanalytical method was developed and validated to study pharmacokinetics of exemestane. The method was based on liquid-liquid extraction of exemestane with methyl t-butyl ether followed by reversed-phase liquid chromatography. Positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode was applied for detection of exemestane. Anastrozole was used as internal standard. Calibration curve, fitted to 1/x2 weighted linear regression model, was linear in the range of 0.1-40.0 ng/mL. Intra-run precision and accuracy were 1.80-3.17% and 103.4-111.5%, respectively. Inter-run precision and accuracy measured within 3 days were 3.37-4.19% and 101.8-109.6%, respectively. Extraction recoveries of exemestane and internal standard were 79.7-86.2% and 82.9-83.6%, respectively. The method was fully validated and may be applied to pharmacokinetic studies in humans after a single dose administration of 25mg exemestane tablets.     

Development and validation of a liquid chromatography/tandem mass spectrometry method for the determination of DMXAA in human and mouse plasma

A rapid, sensitive, and specific LC/MS/MS-based method was developed for determining the concentration of DMXAA in human and mouse plasma. Sample preparation involved a single protein precipitation step using acetonitrile. Separation of DMXAA and 6-isopropoxy-9-oxoxanthene-2-carboxylic acid, the internal standard, was achieved on a Waters X-Terra C(18) (50 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (55:45, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 5-3000 ng/mL. The values for precision and accuracy were <9.6%, except at the LLOQ (5 ng/mL) level, which was within 16.8%. Recovery of DMXAA in mouse plasma was >65%. DMXAA was stable through 2 freeze/thaw cycles, to 2h in mouse plasma or 50% acetonitrile, and on the autosampler to 5.1h. This method was subsequently used to measure concentrations of DMXAA in mice following intraperitoneal administration.     

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of valsartan and hydrochlorothiazide in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of valsartan and hydrochlorothiazide in human plasma. After a simple protein precipitation using acetonitrile, the analytes were separated on a Zorbax SB-Aq C18 column using acetonitrile-10mM ammonium acetate (60:40, v/v, pH 4.5) as mobile phase at a flow rate of 1.2 mL/min. Valsartan and hydrochlorothiazide were eluted at 2.08 min and 1.50 min, respectively, ionized using ESI source, and then detected by multiple reaction monitoring (MRM) mode. The precursor to product ion transitions of m/z 434.2-350.2 and m/z 295.9-268.9 were used to quantify valsartan and hydrochlorothiazide, respectively. The method was linear in the concentration range of 4-3600 ng/mL for valsartan and 1-900 ng/mL for hydrochlorothiazide. The method was successfully employed in a pharmacokinetic study after an oral administration of a dispersible tablet containing 80 mg valsartan and 12.5 mg hydrochlorothiazide to each of the 20 healthy volunteers.     

Development and validation of an HPLC-UV method for iodixanol quantification in human plasma

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.     

Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

Simultaneous quantification of carbamate insecticides in human plasma by liquid chromatography/tandem mass spectrometry

Carbofuran (CFN), carbosulfan (CSN) and fenobucarb (FBC) are carbamate pesticides that are widely used in gardening and agriculture for the control of insects. Human poisoning due to occupational or self-poisoning exposures is also reported, so assays are required to quantify the plasma concentration of these insecticides. An LC-MS/MS method was developed and validated for the simultaneous quantification of these three carbamate insecticides in the plasma of patients with acute intentional self-poisoning. Plasma samples were pretreated by acetonitrile for protein precipitation. Chromatography was carried out on a Luna C18(2) analytical column with gradient elution using a mobile phase containing acetonitrile and water with 10mM ammonium acetate. Mass spectrometric analysis was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) source in the positive ion mode. The total run time was 7 min. The assay was validated over a concentration range from 10 to 1000 ng/ml for CSN and FBC and 20-2000 ng/ml for CFN. The precision and accuracy for both intra- and inter-day determination of all analytes were acceptable (<15%). No significant matrix effect was observed. Stability of compounds was established for short term bench and autosampler storage as well as freeze/thaw cycles. The method was effectively applied to 270 clinical samples from patients with a history of acute intentional carbamate self-poisoning.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of opiorphin in human saliva

Opiorphin, QRFSR-peptide, is a mature product of the PROL1 (proline rich, lacrimal 1) protein that showed beneficial effects in pain management, antidepressant-like actions as well as involvement in colonic motility and erectile physiology. Using opiorphin as a potential biomarker of different pathological states requires the development of robust and sensitive methods. We report a highly sensitive and specific liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the analysis of opiorphin in human saliva. Quantification was based on multiple reaction monitoring using characteristic transitions (m/z 347/120 - as quantifying ion; 347/175 and 347/268 as qualifying ions). The assay was linear in the range of 0-110 ng/ml and the lower limit of quantification reached was 1.0 ng/ml. The intra-day precision and accuracy were between 2.7-5.6% and -2.3 to 3.2%, respectively. The inter-day precision and accuracy were between 10.8-13.7% and -11.0 to 52%, respectively. Mean recovery was 106% and mean matrix effect was 0.97. Opiorphin in TFA treated saliva samples was stable for at least 12h at room temperature and up to 30 days at -20°C. Opiorphin levels in human saliva samples collected from young healthy individuals ranged from 2.8 to 25.9 ng/ml.     

Liquid chromatography-negative ion electrospray tandem mass spectrometry method for the quantification of tacrolimus in human plasma and its bioanalytical applications

A simple, rapid, novel and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of tacrolimus (I) in human plasma, a narrow therapeutic index, potent macrolide immunosuppressive drug. The analyte and internal standard (tamsulosin (II)) were extracted by liquid-liquid extraction with t-butylmethylether using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra ODS column with a mobile phase of 99% methanol and 1% 10mM ammonium acetate buffer. The deprotonate of analyte was quantitated in negative ionization by multiple reaction monitoring (MRM) with a mass spectrometer. The mass transitions m/z 802.5-->560.3 and m/z 407.2-->151.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.05-25ng/ml for tacrolimus in human plasma. The lower limit of quantitation was 50pg/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 2min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in comparative bioavailability studies. The tacrolimus plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration (C(max)) of tacrolimus (5mg oral dose) is 440pg/ml, time to observed maximum plasma concentration (T(max)) is 2.5h and elimination half-life (T(1/2)) is 21h.     

Development and validation of a stereoselective liquid chromatography-tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma

A stereoselective liquid chromatography-tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5-50 microg/L in human plasma. Metoprolol was extracted from plasma by liquid-liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268-->191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r(2)>or=0.9995) over the range 0.5-50 microg/L in plasma, with 0.5 microg/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92-105%.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum

A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-carbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) and separated in less than 3 min on a C18 reverse-phase column using an isocratic elution. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78-50 mg/L for MHD and 0.078-5.0 mg/L for OXC and DHD), showed correlation coefficients (r) better than 0.997. Within day and between days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard. No detectable carry-over and no relevant cross-talk and matrix effect occurred. Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r=0.88) in comparison to our routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies.     

Liquid chromatography/negative ion electrospray tandem mass spectrometry method for the quantification of rosuvastatin in human plasma: application to a pharmacokinetic study

A sensitive liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of rosuvastatin in human plasma. The plasma samples were prepared using liquid-liquid extraction with ethyl ether. Chromatographic separation was accomplished on a Zorbax XDB-C18 (150 mm x 4.6 mm i.d., 5 microm) column. The mobile phase consisted of methanol-water (75:25, v/v, adjusted to pH 6 by aqueous ammonia). Detection of rosuvastatin and the internal standard (IS) hydrochlorothiazide was achieved by ESI MS/MS in the negative ion mode. The lower limit of quantification was 0.020 ng/ml by using 200 microl aliquots of plasma. The linear range of the method was from 0.020 to 60.0 ng/ml. The intra- and inter-day precisions were lower than 8.5% in terms of relative standard deviation (RSD), and the accuracy was within -0.3 to 1.9% in terms of relative error (RE). Compared with the existing methods, the validated method offered increased sensitivity. The method was successfully applied for the evaluation of pharmacokinetics of rosuvastatin after single oral doses of 5, 10 and 20 mg rosuvastatin to 10 healthy volunteers.     

Method development and validation for quantitative determination of methadone enantiomers in human plasma by liquid chromatography/tandem mass spectrometry

A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.     

Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane-isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C(8) micro-bore column (250 mm x 2.1 mm i.d.; 3.5 microm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min(-1). The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml(-1) (r(2) > 0.996). The limit of quantitation was 0.50 ng ml(-1). Accuracy and precision were below the acceptance limits of 15%.     

Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay for the determination of sanfetrinem in human plasma

A rapid, selective and accurate high-performance liquid chromatography–tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 μl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C₁₈(2), 50×2.0 (5 μm) column with a mobile phase consisting of water–acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 μg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma

A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.     

Development and validation of high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma

As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC-MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm x 0.5 mm 50 microm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm x 2.1 mm 5 microm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.     

Development and validation of a liquid chromatography/tandem mass spectrometry method for quantitative determination of amoxicillin in bovine muscle

A simple, quick and economical liquid chromatographic/tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of amoxicillin in bovine muscle was developed and validated. The sample preparation procedure involved a liquid extraction with water, followed by a protein precipitation step with acetonitrile. The extract was purified by a liquid-liquid partition with dichloromethane and the upper aqueous layer was directly injected into the LC-MS/MS system. Chromatographic separation was achieved on a reversed phase column, using a mixture of acetonitrile, water and 0.005% formic acid in water as mobile phase. Gradient elution was performed at a flow rate of 0.2 mL min?1. Amoxicillin was detected using positive electrospray ionization in selected reaction monitoring (SRM) mode and was quantified using terbutaline as internal standard. The responses for standards prepared in solvent and in matrix were equivalent and additionally the absence of signal suppression was confirmed by the post column infusion technique. Amoxicillin stability in standard solution and in matrix was investigated at different times and storage conditions. Amoxicillin standards prepared in water were stable on storage up to 20 days at -20°C. Amoxicillin stability in matrix (spiked bovine muscle samples) was assessed up to 15 days at -20°C. The method was validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, linearity, trueness, precision, decision limit (CCα) and detection capability (CCβ). All the trueness values fell within a range between 14.5% and 6.3%. Precision values for all levels of concentration tested were lower than the relative limit calculated by the Horwitz equation. The amoxicillin MRL is set at 50 μg kg?1 and the CCα and CCβ of the method were 61.2 μg kg?1 and 72.4 μg kg?1, respectively.