Development and validation of highly sensitive method for determination of misoprostol free acid in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A highly sensitive, selective and evaporation free SPE extraction, ESI-LC-MS/MS method has been developed for estimation of misoprostol free acid in human plasma using misoprostol acid-d(5) as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M-H] anions, m/z 367-249 for misoprostol acid and m/z 372-249 for the IS. The total run time was 5.0 min and the elution of misoprostol acid and misoprostol acid-d(5) (IS) occurred at 3.6 min. The developed method was validated in human plasma with a lower limit of quantification of 2.5 pg/mL. A linear response function was established for the range of concentrations 2.5-1200 pg/mL (r>0.998) for misoprostol acid in human plasma. The intra and inter-day precision values for misoprostol acid met the acceptance as per FDA guidelines. Misoprostol acid was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.     

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.     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of scutellarin in human plasma: Application to a pharmacokinetic study

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of scutellarin in human plasma has been developed. Samples were prepared using solid phase extraction and analyzed on a C(18) column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol-water (0.1% formic acid), using gradient procedure. The analyte and internal standard baicalin were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.2-20.0 ng/mL. The lower limit of quantification (LLOQ) was 0.2 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.4%. The accuracy determined at three concentrations (1.0, 5.0 and 10.0 ng/mL for scutellarin) was within +/-5.0% in terms of relative error. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of scutellarin guttate pills in 20 healthy volunteers.     

Rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of fosfomycin in human plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for determination of fosfomycin was developed and validated. Following protein-precipitation, the analyte and internal standard (fudosteine) were separated from human plasma using an isocratic mobile phase on an Ultimate XB-CN column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 137-->79 and m/z 178-->91 was performed to quantify fosfomycin and fudosteine, respectively. The method was linear in the concentration range of 0.10-12.0 microg/mL using 50 microL of plasma. The lower limit of quantification was 0.10 microg/mL. The intra- and inter-day relative standard deviation over the entire concentration range was less than 10.6%. Accuracy determined at three concentrations (0.25, 1.00 and 8.00 microg/mL for fosfomycin) ranged from -1.0% to -4.2% in terms of relative error. Each plasma sample was chromatographed within 5.0 min. The method was successfully used in a bioequivalence study of fosfomycin in human plasma after an oral administration of capsules containing 1.0 g fosfomycin (approximately 1.3g calcium fosfomycin).     

A capillary liquid chromatographic/tandem mass spectrometric method for the quantification of gamma-aminobutyric acid in human plasma and cerebrospinal fluid

A sensitive and reliable method for the determination of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in human plasma and cerebrospinal fluid (CSF) has been developed. The method is based on capillary liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using deuterium-labeled GABA (gamma-aminobutyric acid-2,2-D(2), GABA-d(2)) as internal standard. Pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) was deployed, allowing both effective in-line pre-concentration and sensitive tandem MS detection of the analyte. An extraction column (10 mm x 0.25 mm, 7 microm, C(18)) was used for preconcentrating and stacking the sample. Separation was carried out on an analytical column (50 mm x 0.25 mm, 5 microm, C(18)). Characteristic precursor-to-product ion transitions, m/z 267--> 249 (for NBD-GABA) and m/z 269--> 251 (for NBD-GABA-d(2)) were monitored for the quantification. A linear calibration curve from 10 to 250 ng/mL GABA with an r(2) value of 0.9994 was obtained. Detection limit was estimated to be 5.00 ng/mL GABA (S/N = 3). Human plasma and CSF samples were analyzed. The concentrations of GABA were found to be 98.6 +/- 33.9 ng/mL (mean +/- S.D., n = 12), and 44.3 +/- 10.0 ng/mL (n = 6) in plasma and CSF, respectively.     

Determination of dimenhydrinate in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a relative bioavailability study

Here we present a sensitive and specific liquid chromatography-tandem mass spectrometric method for the quantification of dimenhydrinate (I) in human plasma. Sample preparation is conducted using citalopram (II) addition as an internal standard (IS), liquid-liquid extraction with basified plasma using a mixture hexane/acetate (1:1, v/v) as the extracting solvent, and the final extract reconstituted in the mobile phase. I and II (IS) were injected in a C8 column with the mobile phase composed of methanol:isopropanol:water:formic acid (78.00:19.92:2.00:0.08, v/v/v/v) and monitored using a positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 256.0>167.0 and m/z 325.0>109.0 for I and II, respectively. The limit of quantification (LOQ) was 0.4 ng/mL, the dynamic range being 0.4-200 ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of plasma samples taken up to 24 h after oral administration of 100 mg of dimenhydrinate in healthy volunteers demonstrated its applicability to bioavailability studies.     

Gas chromatographic–tandem mass spectrometric determination of acetylsalicylic acid in human plasma after oral administration of low-dose aspirin and guaimesal

A fully validated gas chromatographic–tandem mass spectrometric (GC–MS–MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[²H₃]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-μl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC–MS–MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0–1 μg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0–100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.     

Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma

A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 microL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 mm x 2.1 mm Agilent Zorbax SB-phenyl 5 microm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 microL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3>541.3, 544.2>501.2, and 615.3>572.3, respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 min, respectively, with a total run time of 7 min. The assay was linear over the range 0.5-3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 microL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high-performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to pharmacokinetic study of nalmefene in humans

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for quantification of nalmefene in human plasma. An aliquot of 200 microL plasma sample was simply precipitated by 400 microL methanol. Separation of nalmefene and the internal standard hydromorphone from the interferences was achieved on a C(18) column followed by MS/MS detection. The analytes were monitored in the positive ionization mode with a TurboIonspray source. The method had a total chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 10-5000 pg/mL. The lower limit of quantification (LLOQ) was 10 pg/mL. The intra- and inter-day precision was less than 10.1% determined from QC samples at concentrations of 30, 300 and 4500 pg/mL, and the accuracy was within +/-3.4%. As the method was more sensitive (10 times higher) than those reported previously, we investigated the pharmacokinetics of nalmefene in healthy volunteers after a single intravenous injection of low dose (30 microg) of nalmefene hydrochloride for the first time.     

Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10-30microL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20microL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi Polar RP analytical column (50mmx3.0mm, 4microm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441-->175 for A and 469-->175 for B, respectively. The assays were validated over the concentration range of 0.5-200ng/mL for human plasma and CSF. Replicate analyses (n=5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.     

Plasma and cerebral spinal fluid tranexamic acid quantitation in cardiopulmonary bypass patients

A method for the determination of tranexamic acid (TXA) in human plasma and cerebral spinal fluid (CSF) was developed. Analyses were performed by ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC–MS/MS) using ?-aminocaproic acid (ACA) as an internal standard. TXA and ACA were extracted from a 50 μL sample of plasma or CSF using a methanol protein crash protocol, and chromatographic separation was performed on an ACQUITY? TQD mass spectrometer using a UPLC C18 BEH 1.7 μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation was performed by positive ion electrospray ionization using the multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1–10.0 μg/mL, with lower limit of quantitation of 0.1 μg/mL for TXA. The intra- and inter-assay precision was less than 12% and 13% respectively at the plasma and CSF TXA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around-time. The method has been successfully applied to assess the plasma and CSF concentrations of tranexamic acid achieved with only one dosing regimen of tranexamic acid in patients undergoing cardiopulmonary bypass surgery (CPB).     

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 bioanalytical methods for Imidafenacin (KRP-197/ONO-8025) and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

Imidafenacin (KRP-197/ONO-8025, IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a new antimuscarinic agent currently under application for the indication of treatment of overactive bladder in Japan. We developed and validated the sensitive and selective bioanalytical methods for the extremely low levels of IM and its metabolite, M-2 (Method 1), M-4 (Method 2) and M-9 (Method 3) in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In each method, plasma sample was extracted by solid phase extraction, separated on a semi-micro high performance liquid chromatography column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization or ionspray interface. Selected reaction monitoring mode was used for quantification. Each method was found to have acceptable accuracy, precision, stability, selectivity and linearity over the concentration range of 10-500 pg/mL for IM and M-2, 10-1000 pg/mL for M-4 and 50-5000 pg/mL for M-9. Using these analytical methods, concentration profiles of IM and its metabolites in human plasma were successfully determined even in the low pg/mL levels after oral administration of IM at the therapeutic dosage of 0.1 mg.     

A rapid and robust liquid chromatography/tandem mass spectrometry method for simultaneous analysis of anti-tuberculosis drugs—Ethambutol and pyrazinamide in human plasma

Ethambutol and pyrazinamide are two first-line anti-tuberculosis drugs. Though they are normally combined for the treatment, their highly different polarity complicates simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of these two drugs in human plasma with decent peak shape and retention. Here we report a rapid and robust LC/MS/MS method for the simultaneous determination of these two drugs in human plasma. Human plasma samples, together with the isotopically labeled internal standards were extracted using protein precipitation, and then separated on a Chromolith SpeedROD RP-18e column and detected with mass spectrometry. The mobile phase is 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol. Addition of trifluoroacetic acid in the mobile phases was found to be able to improve peak shape as well as to increase the retention of ethambutol, thus being able to analyze these two drugs at the same time with both drugs having decent peak shape and enough retention on a C₁₈ column. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The standard curve range was 10.0–5000 ng/mL for ethambutol and 50.0–25,000 ng/mL for pyrazinamide using a plasma sample volume of 50.0 μL. This method has a very short run time of 3.8 min. The method has been fully validated, and <15% relative standard deviation was obtained for both analytes.     

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

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.     

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.     

Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry

The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.