Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies

Oxidation of methionine residues and deamidation of asparagine residues are the major causes of chemical degradation of biological pharmaceuticals. The mechanism of these non-enzymatic chemical reactions has been studied in great detail. However, the identification and quantification of oxidation and deamidation sites in a given protein still remains a challenge. In this study, we identified and characterized several oxidation and deamidation sites in a rat/mouse hybrid antibody. We evaluated the effects of the sample preparation on oxidation and deamidation levels and optimized the peptide mapping method to minimize oxidation and deamidation artifacts. Out of a total number of 18 methionine residues, we identified six methionine residues most susceptible to oxidation. We determined the oxidation rate of the six methionine residues using 0.05% H₂O₂ at different temperatures. Methionine residue 256 of the mouse heavy chain showed the fastest rate of oxidation under those conditions with a half life of approximately 200 min at 4 °C and 27 min at 37 °C. We identified five asparagine residues prone to deamidation under accelerated conditions of pH 8.6 at 37 °C. Kinetic characterization of the deamidation sites showed that asparagine residue 218 of the rat heavy chain exhibited the fastest rate of deamidation with a half live of 1.5 days at pH 8.6 and 37 °C. Analysis of antibody isoforms using free flow electrophoresis showed that deamidation is the major cause of the charged variants of this rat/mouse hybrid antibody.     

Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method

A rapid 2 min liquid chromatography–tandem mass spectrometry (LC–MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1–5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm × 2.0 mm i.d.) column at 50 °C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC–MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

A novel validated procedure for the determination of nicotine,eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R²) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (?20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.     

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography–tandem mass spectrometry

A rapid liquid chromatographic–tandem mass spectrometric (LC–MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC–MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 μg L^?1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC–MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) were found to be below the chosen validation level of 1 μg L^?1 for all compounds.     

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology

In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 μL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard? column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters^® Quattro Premier? XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9 > 145.6 and 193.9 > 147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97–113%) and the method showed good linearity to 10,000 nmol/L (r² = 0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC–MS/MS and HPLC showed good agreement between the two methods but this LC–MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.     

Deamidation of peptides in aerobic nitric oxide solution by a nitrosative pathway

Hydrolytic deamidation of asparagine (Asn) and glutamine (Gln) residues to aspartate (Asp) and glutamate (Glu), respectively, can have significant biological consequences. We hypothesize that a wholly different mechanism of deamidation might occur in the presence of aerobic nitric oxide (NO). Accordingly, we examined the deamidating ability of aerobic NO toward three model peptides, 2,4-dinitrophenyl (DNP)-Pro-Gln-Gly, Lys-Trp-Asp-Asn-Gln, and Ser-Glu-Asn-Tyr-Pro-Ile-Val, incubating them with the NO-generating compound, Et₂N[N(O)NO]Na (DEA/NO, 30–48 mM), in aerobic, pH 7.4, buffer at 37 °C. DNP-Pro-Glu-Gly was detected after 2 h, while Lys-Trp-Asp-Asp-Gln, Lys-Trp-Asp-Asn-Glu, and Ser-Glu-Asp-Tyr-Pro-Ile-Val were detected within 10 min, accumulating in apparent yields of up to ∼10%. In the latter case, tyrosine nitration was also observed, producing the expected nitrotyrosine residue. DEA/NO solutions preincubated to exhaust the NO before the peptides were added did not induce detectable deamidation. The data demonstrate that aerobic NO exposures can lead to nitrosative deamidation of peptides, a pathway that differs from the established hydrolytic deamidation mechanism in several key respects: the by-products of the former are molecular nitrogen and an acid (HONO) while that of the latter is a base (NH₃); the nitrosative path can in principle proceed in the absence of water molecules; Asn is much more easily deamidated than Gln in the hydrolytic pathway, while the two amino acid residues were deamidated to a similar extent by exposure to NO in the presence of oxygen.     

Quantitative determination of BAF312, a S1P-R modulator,in human urine by LC–MS/MS: Prevention and recovery of lost analyte due to container surface adsorption

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC–MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the ‘non-specific loss’ of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC–MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the ‘non-specific binding’ of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. β-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of β-cyclodextrin to untreated urine samples does not recover the ‘lost’ analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200–20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from ?3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid–liquid extraction in combination with an isocratic LC separation was employed. The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at ≤?60 °C and for at least 3 months when stored at ≤?60 °C. The current work could serve as a simple example in trouble shooting non-specific binding or container surface adsorption in quantitative analysis of urine samples.     

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.     

A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D

BackgroundThe accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D₂ and 25OH-D₃ in serum. The new method was compared with two widely used commercially available immunoassays.MethodsSample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.ResultsUsing 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D₂ and 25OH-D₃ with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r = 0.931; LC Tandem MS vs. ECLIA: r = 0.784; RIA vs. ECLIA: r = 0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.ConclusionThe new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Purification,characterization and mass spectrometric identification of two thermophilic xylanases from Sporotrichum thermophile

Two xylanases were purified to electrophoretic homogeneity from the thermophilic fungus Sporotrichum thermophile grown in a submerged liquid culture using wheat straw as carbon source. The enzymes, StXyn1 and StXyn2, have molecular masses of 24 kDa and 48 kDa, respectively, and are optimally active at pH 5 and at 60 °C. Both enzymes displayed remarkable stability up to 50 °C for 1 h, exhibiting a half-life of 60 min (StXyn1) and 115 min (StXyn2) at 60 °C. Biochemical characterization of the two xylanases against poly- and oligosaccharides indicated that StXyn1 and StXyn2 hydrolytic profiles match those of xylanase family 11 and family 10, respectively. LC–MS/MS analysis provided peptide mass and sequence information that assisted the identification of the corresponding xylanase genes from the S. thermophile genome and the classification of the two purified StXyn1 and StXyn2 as a family GH11 and GH10 endo-1,4-β-xylanases, respectively.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Simultaneous analysis of isomers of escin saponins in human plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study after oral administration

A rapid and sensitive bioassay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of four isomeric escin saponins (escin Ia, escin Ib, isoescin Ia and isoescin Ib) in human plasma has been developed and validated. Sample preparation of plasma after addition of telmisartan as internal standard (I.S.) involved solid-phase extraction (SPE) on C18 cartridges. Separation was based on reversed phase chromatography using gradient elution with methanol–acetonitrile (50:50, v/v) and 10 mM ammonium acetate solution (pH 6.8). MS/MS detection in the positive ion mode used multiple reaction monitoring of the transition at m/z 1113.8 → 807.6. Stability issues with the four saponins required the addition of formic acid to plasma samples prior to storage at ?80 °C and analysis within 30 days. The method was linear at concentrations up to 10 ng/mL with correlation coefficients > 0.996 for all analytes. The lower limit of quantitation (LLOQ) for all four saponins was 33 pg/mL. Intra- and inter-day precisions (as relative standard deviation) were all <15% and accuracies (as relative error) in the range ?5.3% to 6.1%. The method was successfully applied to a pharmacokinetic study of escins in healthy volunteers after oral administration of sodium aescinate tablets containing 60 mg escin saponins.     

Quantitation of bergenin in human plasma by liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an assay for quantitation of bergenin in human plasma using liquid chromatography/tandem mass spectrometry (LC–MS/MS). Bergenin and the internal standard (I.S.), 5-bromo-2,4(1H,3H)-pyrimidinedione (5-BrU), were separated by reversed phase HPLC and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M?H]^? MRM transition of bergenin at m/z 326.9 → 312.3 was used for quantitation and the transition at m/z 188.9 → 42.2 was used to monitor 5-BrU. Stability issues with bergenin required the addition of ascorbic acid to plasma samples prior to storage and analysis within 10 days storage at ?80 °C. The method was linear in the range 3–1000 ng/mL with intra- and inter-day precision of 3.94–5.96 and 1.62–8.31%, respectively, and accuracy <2.33%. The assay was successfully applied to a pharmacokinetic study in healthy volunteers after administration of a single 250 mg oral dose.     

Quantitation of polar analytes using column-switching: Application to oxycodone and three metabolites in human plasma

We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10–12 μm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 × 50 mm, 3.5 μm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 μl, quantification ranges were 25–10,000 pg/ml for OXM and NOXM and 50–10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.     

Validated liquid chromatography–tandem mass spectrometry method for quantitative determination of dauricine in human plasma and its application to pharmacokinetic study

A highly sensitive and selective LC–MS/MS method was developed and validated for the determination of dauricine in human plasma, using protopine as internal standard (IS). The analyte and IS were extracted by liquid–liquid extraction and analyzed by LC–MS/MS. Chromatographic separation was performed on Agilent TC-C₁₈ column with a mobile phase of methanol–water–glacial acetic acid (60:40:0.8, v/v/v) at a flow rate of 0.7 mL/min. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The method was linear over the concentration range of 1–200 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL in human plasma with acceptable precision and accuracy. The intra- and inter-day precision was less than 5.9% determined from quality control (QC) samples at concentrations of 2.0, 20.0 and 160 ng/mL, and the accuracy was within ±9.9%. This method was successfully applied for the evaluation of pharmacokinetics of dauricine after oral doses of 100, 300 and 600 mg phenolic alkaloids of menispermum dauricum tablet (PAMDT) to 12 Chinese healthy volunteers.     

Simple and selective method for the determination of various tyrosine kinase inhibitors used in the clinical setting by liquid chromatography tandem mass spectrometry

A fast, sensitive, universal and accurate method for the determination of four different tyrosine kinase inhibitors from biological material was developed using LC–MS/MS techniques. Utilizing a simple protein precipitation with acetonitrile a 20 μl sample volume of biological matrixes can be extracted at 4 °C with minimal effort. After centrifugation the sample extract is introduced directly onto the LC–MS/MS system without further clean-up and assayed across a linear range of 1–4000 ng/ml. Chromatography was performed using a Dionex Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0 mm × 100 mm, 3 μm) column and eluted at 200 μl/min with a tertiary mobile phase consisting of 20 mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 μl to 1 μl depending on the concentration of the drug observed. Samples were observed to be stable for a maximum of 48 h after extraction when kept at 4 °C. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for Gefitinib (447.1 m/z; 127.9 m/z), Erlotinib (393.9 m/z; 278.2 m/z), Sunitinib (399.1 m/z; 283.1 m/z) and Sorafenib (465.0 m/z; 251.9 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantification (LOQ) from cell culture medium were as follows: Gefitinib: 100.2 ± 3.8%, 11.2 nM; Erlotinib: 101.6 ± 3.7%, 12.7 nM; Sunitinib: 100.8 ± 4.3%, 12.6 nM; Sorafenib: 93.9 ± 3.0%, 10.8 nM, respectively. This was reproducible for plasma, whole blood, and serum. The method was observed to be linear between the LOQ and 4000 ng/ml for each analyte. Effectiveness of the method is illustrated with the analysis of samples from a cellular accumulation investigation and from determination of steady state concentrations in clinically treated patients.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.