Development and validation of Lenalidomide in human plasma by LC-MS/MS

A highly sensitive and ultra-fast high performance liquid chromatography- tandem mass spectrometry (LC–MS/MS) assay is developed and validated for the quantification of Lenalidomide in human plasma. Lenalidomide is extracted from human plasma by Liquid- Liquid Extraction by Ethyl Acetate and analyzed using a reversed phase isocratic elution on a XTerra RP18, (4.6 × 50 mM, 5 µm) column. A 0.1% Formic acid: Methanol (10:90% v/v), is used as mobile phase and detection was performed by Triple quadrupole mass spectrometry LC-MS/MS using electrospray ionization in positive mode. Fluconazole is used as the internal standard. The lower limit of quantification is 9.999 ng/mL for Lenalidomide. The calibration curves are consistently accurate and precise over the concentration range of 9.999 to 1010.011 ng/mL in plasma for Lenalidomide. This novel LC–MS/MS method competes with all the regulatory requirements and shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic and bioequivalence studies in humans.     

Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.0 mm I.D., 5 microm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 microL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.     

Determination of SCH 211803 by nanoelectrospray infusion mass spectrometry: evaluation of matrix effect and comparison with liquid chromatography-tandem mass spectrometry

A high throughput assay for SCH 211803, an M2 muscarinic receptor antagonist in human plasma using nanoelectrospray infusion tandem mass spectrometry is described. Sample processing consisted of protein precipitation followed by solid phase extraction using octadecasilyl resin-filled pipette tips on a liquid handling robotic system. The sample extracts were infused directly to the mass spectrometer using a nanoelectrospray interface in a silicon chip format. SCH 211803 was quantified in plasma over the concentration range of 1-1000 ng/mL. In comparison with a liquid chromatography-tandem mass spectrometry assay, the nanoelectrospray method has comparable accuracy, precision and limit of quantitation, with a nine-fold improvement in sample throughput. Using the nanoelectrospray assay, ion suppression was evaluated and found to be 15%. This represented a four-fold reduction in matrix suppression when compared to a conventional electrospray source operating in the flow injection analysis mode at a flow rate common for LC-MS/MS analysis.     

Quantification of dimethindene in plasma by gas chromatography—mass fragmentography using ammonia chemical ionization

A gas chromatographic—mass fragmentographic method using ammonia chemical ionization for the determination of dimethindene in human plasma is described. The drug was isolated from plasma by liquid—liquid extraction with hexane—2-methylbutanol. Plasma components were separated on a capillary column coated with chemically bonded methyl silicone. For detection of dimethindene, its quasi-molecular ion (M + H⁺) was mass fragmentographically monitored after chemical ionization with ammonia as reagent gas. Dimethindene was quantified using methaqualone as the internal standard: the quantification limit in plasma was 0.2 ng/ml, the within-run precision was 8.0% and the inter-run precision 5.6%. The plasma concentration—time profile was established after a single dose of 4 mg of dimethindene with an average maximum concentration of 5.5 ng/ml, detectable up to 48 h post application.     

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.     

Determination of etoricoxib in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionisation

The validation of a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of the selective cyclooxygenase-2 inhibitor etoricoxib in human plasma with phenazone as internal standard is described. The plasma samples were extracted by solid-phase extraction using polymer-based cartridges. Chromatography was carried out on a short, narrow bore RP C(18) column (30x2 mm). Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2-->280.2 and m/z 189.0-->104.1. The analytical method was validated over the concentration range 0.2-200 ng/ml. The limit of quantification was 0.2 ng/ml. The method is applicable to pharmacokinetic studies in humans.     

Determination of isoniazid and its hydrazino metabolites, acetylisoniazid, acetylhydrazine, and diacetylhydrazine in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric assay for isoniazid and its hydrazino metabolites in human plasma was developed. The trimethylsilyl derivatives of diacetylhydrazine and acetylisoniazid and of the benzaldehyde hydrazones of acetylhydrazine and isoniazid were separated on a 1% OV-17 column and quantitated by single ion monitoring using a LKB 9000 mass spectrometer. Deuterated analogues served as internal standards. The method is well suited for the determination of the hepatotoxic hydrazino metabolites of isoniazid in human plasma following an oral therapeutic dose of isoniazid.     

Measurement of succinylcholine concentration in human plasma by electrospray tandem mass spectrometry

An electrospray mass spectrometric method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine (SUX) is described. An extraction method compatible with direct infusion inlet was developed and leads to an analysis cycle time of 7--8 min instead of 25 min that would be required for HPLC inlet. SUX was extracted from human plasma on C1 solid-phase cartridges and was analyzed using positive ion electrospray tandem mass spectrometry (ESI-MS/MS). SUX plasma concentrations were determined by a stable isotope dilution assay using hexadeuterosuccinylcholine diiodide (SUXd6) as the internal standard. The calibration curve was prepared using the ratio of intensities of the major product ions in the collision-induced dissociation spectrum for known concentration ratios of SUX and SUXd6 in plasma. Calibration curves for the quantification were linear from 25 to 4000 ng/ml. For intraday precision, CV were < or =6% and accuracy ranged from 98 to 103%. For the interday precision, CV were < or =10% and accuracy ranged from 90 to 102%. This method is specific, sensitive, reproducible, and practical in a clinical setting.     

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.     

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).     

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.     

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

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

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Determination of rimonabant in human plasma and hair by liquid chromatography-mass spectrometry

Rimonabant is the first therapeutically relevant cannabinoid antagonist, licensed in Europe for treatment of obesity when a risk factor is associated. The objective of this study was to develop and validate a method for measurement of rimonabant in human plasma and hair using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Rimonabant and AM-251 (internal standard) were extracted from 50muL of plasma or 10mg of hair using diethylether. Chromatography was performed on a 150mmx2.1mm C18 column using a mobile phase constituted of formate buffer/acetonitrile. Rimonabant was ionized by electrospray in positive mode, followed by detection with mass spectrometry. Data were collected either in full-scan MS or in full-scan MS/MS mode, selecting the ion m/z 463.1 for rimonabant and m/z 555.1 for IS. The most intense product ion of rimonabant (m/z 380.9) and IS (m/z 472.8) were used for quantification. Calibration curves covered a range from 2.5 (lower limit of quantification) to 1000.0ng/mL (upper limit of quantification) in plasma and from 2.5 to 1000.0pg/mg in hair. Validation results demonstrated that rimonabant could be accurately and precisely quantified in both matrixes: accuracy and precision were within 85-115% and within 15% of standard deviation, respectively. Stability studies in plasma showed that rimonabant was stable during the assay procedure, but a 30% decrease was observed for one concentration after 3 weeks at -20 degrees C. This simple and robust LC-MS/MS method can be used for measuring rimonabant concentrations in human plasma and hair either in clinical or in forensic toxicology.     

Determination of carboplatin in plasma and tumor by high-performance liquid chromatography-mass spectrometry

Carboplatin is a platinum analogue that is used in a number of chemotherapeutic regimens for solid tumors, such as lung and ovarian carcinomas. Most often characterization of carboplatin's pharmacokinetic properties is based on measurement of platinum, rather than intact carboplatin. We have developed a sensitive LC-MS method for the determination of intact carboplatin in plasma ultrafiltrate and in tumor tissue. Carboplatin was extracted from rat plasma ultrafiltrate and tumor samples using solid-phase extraction cartridges and analyzed using reversed-phase chromatography with positive electrospray ionization followed by mass spectrometric detection. Using 50 microliter of plasma ultrafiltrate or 140 microliter of tumor homogenate supernatant, the extraction afforded a recovery of 58.7 and 45.8% for plasma and tumor, respectively. The mobile phase was 5% acetonitrile in 0.5% acetic acid at 0.2 ml/min that yielded a retention time of carboplatin of 2.2 min. The method has been validated at carboplatin plasma ultrafiltrate concentrations from 0.07 to 2.5 microgram/ml, and from 0.03 to 1.3 microgram/ml in tumor homogenates. The main advantages of this method compared with earlier methods are the ability to measure intact carboplatin in a sensitive and specific manner.     

Simultaneous determination of gemcitabine and gemcitabine-squalene by liquid chromatography-tandem mass spectrometry in human plasma

Gemcitabine-squalene is a new prodrug that self-organizes in water forming nanoassemblies. It exhibits better anti-cancer properties in vitro and in vivo than gemcitabine. A liquid chromatography/tandem mass spectrometry assay of gemcitabine-squalene and gemcitabine was developed in human plasma in order to quantitate gemcitabine and its squalene conjugate. After protein precipitation with acetonitrile/methanol (90/10, v/v), the compounds were analyzed by reversed-phase high performance liquid chromatography and detected by tandem mass spectrometry using multiple reaction monitoring. The method was linear over the concentration range of 10-10,000 ng/ml of human plasma for both compounds with an accuracy lower than 10.4% and a precision below 14.8%. The method showed a lower limit of quantitation of 10 ng/ml of human plasma for dFdC and dFdC-SQ. A preliminary in vivo study in mice was shown as application of the method as no significant difference between human and mice plasma for the analysis of dFdC and dFdC-SQ was demonstrated.     

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 bufuralol and its major metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bufaralol, a benzofuran analogue, in plasma is described.The unchanged drug, the major metabolites and an internal standard are extracted from plasma, purified by back-extraction steps and thereafter separated using a reversed-phase liquid chromatographic system. The detection is carried out by means of a fluorescence detector and an UV detector connected in series. The sensitivity of the assay for the unchanged drug and the major metabolite is about 1 ng/ml plasma using a 0.5 ml specimen per analysis and the relative standard deviation of the whole assay lies in the range ± 4–5%.The procedure was successfully used to determine plasma levels in volunteers following a single oral dose of 40 mg of bufaralol. The results obtained using the new high-performance liquid chromatographic method were compared with those determined by another method which combines gas chromatography with mass fragmentography, and it was found that these two sets of results coincided quite well.     

Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry

The validation of a method for the determination of mevalonic acid (MVA; after conversion to the lactone, MVAL) in human plasma, using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS-MS), is reported. MVAL and deuterated internal standard were extracted from human plasma samples using automated solid-phase extraction. Analysis was conducted by column-switching, reversed-phase LC-MS-MS, using two hyper-cross-linked styrene-divinylbenzene copolymer sorbent reversed-phase columns. An assay range of 0.2-35 ng/ml and a lower limit of quantitation (LLOQ) of 0.2 ng/ml were achieved with acceptable accuracy and precision. MVA was stable in plasma under a variety of storage conditions.     

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.