Triple Quadrupole Mass Research Articles

Validation of an LC-MS/MS method for the quantitative determination of the orexin receptor antagonist almorexant and its four primary metabolites in human plasma

A sensitive and selective LC-MS/MS method has been developed to quantify almorexant and its four primary metabolites M3, M5, M6, and M8 in human plasma samples. The method involved protein precipitation with acetonitrile in the high calibration range and liquid/liquid extraction with ethyl acetate in the low calibration range. Labeled internal standards were available for four analytes. Separation was performed with an Eclipse XDB-C18 (2.1mm×150mm, particle size 3.5μm) and a XBridge C18 column (2.1mm×50mm, particle size 3.5μm). The mobile phases were mixtures of acetonitrile, methanol, and water containing 1% formic acid; flow rate was 400μL/min. The triple stage quadrupole mass spectrometer was operated in ESI mode and the methods were linear over a range of 0.400-100ng/mL (almorexant, M5, M6), 1.00-100ng/mL (M3, M8), and 50.0-1000ng/mL (all analytes). The inter-day coefficients of variation were equal to or smaller than 10.5%. The inter-day accuracies were between 92.1% and 105.2%. The validated method was successfully applied to the pharmacokinetic assessment of almorexant and its metabolites in several phase I studies.

Development of Mass Spectrometry Selected Reaction Monitoring Method for Quantitation and Pharmacokinetic Study of Stepharine in Rabbit Plasma

Highly sensitive liquid chromatography mass spectrometry method on triple quadrupole (QQQ) mass spectrometer was successfully applied for pharmacokinetic study of stepharine in rabbit plasma. Specific ion transitions of stepharine protonated precursor ion were selected and recorded in the certain retention time employing dynamic selected reaction monitoring mode. The developed method facilitated quantitative measurements of stepharine in plasma samples in linear range of five orders of magnitude with high accuracy and low standard deviation coefficient and pharmacokinetics parameters were calculated. The apparent volume of stepharine distribution (estimated as ratio of clearance to elimination rate constant, data not shown) allows us to assume that stepharine was extensively distributed throughout the body.

A UPLC-MS/MS Method for Qualification of Quercetin-3-O-β- D-glucopyranoside-(4®1)-α-L-rhamnoside in Rat Plasma and Application to Pharmacokinetic Studies

A sensitive and accurate ultra-performance liquid chromatography coupled with triple quadrupole mass (UPLC-MS/MS) method was developed for the determination of quercetin-3-O-β-D-glucopyranoside-(4→1)-α-L-rhamnoside (QGR) in rat plasma using rutin as internal standard. Chromatographic separation was achieved on a Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a gradient elution of acetonitrile and 0.10% formic acid (v/v) at a flow rate of 0.4 mL/min. QGR and rutin were detected using electrospray negative ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. The method demonstrated good linearity and did not show any endogenous interference with the QGR and rutin peaks. This method was successfully applied to a pharmacokinetic study of QGR in rats after intravenous (20 mg/kg) and oral (40 mg/kg) administration, and the results showed that the compound was poorly absorbed, with an absolute bioavailability of approximately 3.41%.

Determination of terpene lactones in Ginkgo biloba leaves in different ages by UPLC-TQ-MS

To establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass (UPLC-TQ-MS) for determination of four terpene lactones. Chromatographic separation was carried out on a ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with isocratic elution of 70% methanol at a flow rate of 0.4 mL x min(-1), the column temperature was set at 30 degrees C; Waters Xevo TQ worked in multiple reaction monitoring mode. All calibration curves were linear (r > 0.990 3) over the tested ranges. The average recoveries ranged from 98.83% to 103.9% with RSD value below 3.0%. The contents of total terpene lactones in Ginkgo biloba leaves were significantly different in different ages. The contents in the leaves of young ginkgo tree were higher than that in old tree. The method was simple and fast with high precision, sensitivity and repeatability, which can be used for qualitative and quantitative analysis of terpene lactones in G. biloba leaves.

Qualitative and Quantitative Analysis of Flavonoids in <i>Humulus scandens</i> (Lour.)Merr. by UPLC-TQD and HPLC

To qualify and quantify the flavonoids existing in Humulus scandens (Lour.)Merr., a Ultra performance liquid chromatography triple-quadrupole tandem mass spectrometer (UPLC-TQD) method was applied to preliminarily analyze the flavonoids exiting in Humulus. And a High performance liquid chromatography (HPLC) method was used to analyze the flavonoids existing in Humulus. Flavonoids in methanol extracts of Humulus were cleaned up by a C18 cartridge, separated by a PLASMA ODS column(5μm,250×4.6mm), with a gradient elution of methanol and 0.1% formic acid at a flow rate of 1 mL•min-1 . Analytes were detected with ultraviolet detector (UV) at 280nm. Flavonoids, including Epicatechin, Luteolin-7-O-Glucoside, Vitexin or Apigenin-7-glucoside, Morin hydrate or Quercetin dehydrate and Luteolin were found in Humulus according to the UPLC-TQD. Results of HPLC analysis showed the linear ranges of 11 flavonoids were 0.1mg•L-1-20mg•L-1 with the correlation coefficient (r) all above 0.9909. Limits of detection (signal/ noise=3) was 100Superscript text μg•L-1. The average recoveries of all the compounds spiked at four levels of 200μg•L-1, 500μg•L-1, 1 mg•L-1 and 1.5 mg•L-1 were in the ranges of 70.8%-112.1%, 74.6%-116.6%, 73.1%-110.6%, 73.7%-99.9%, with the corresponding relative standard deviation (RSD, n=6) of 2.5%-5.8%, 0.8%-4.3%, 0.8%-3.7%, 1.7%-3.8%, respectively. This method ,which was proved to be precise, sensitive and reliable, could be applied to analyze flavonoids in leaves.

Determination of nucleosides and nucleobases in Isatidis Radix by HILIC-UPLC-MS/MS

To establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass (HILIC-UPLC-MS/MS) for determination of sixteen nucleosides and nucleobases in Isatidis Radix. Chromatographic separation was carried out on a Acquity UPLC BEH Amide column with gradient elution of acetonitrile (containing 0.1% acetic acid) and water (containing 0.8% acetic acid and 10 mmol L−1 ammonium acetate) at a flow rate of 0.3 mL min−1, the column temperature was set at 35 °C; Waters Xevo™ TQ worked in multiple reaction monitoring mode. Each component were separating in 11 min. All calibration curves were linear (r2 > 0.997) over the tested ranges. The limits of detection of these compounds were 0.02–42.54 ng mL−1. The limits of quantitation of these compounds were 0.05–98.18 ng mL−1. The average recoveries were in the range of 93.81–105.77% with RSD value less than 7.5%. The result showed that almost all of these Isatidis Radix were rich in nucleosides and nucleobases, but their contents were obviously various. The method was simple and fast with high precision, sensitivity and repeatability, which can be used for determination of this type class of nucleosides and nucleobases in other medicinal herbs.

Determination of Voriconazole in Human Plasma by HPLC–ESI-MS and Application to Pharmacokinetic Study

A fast, sensitive, high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of voriconazole in human plasma. Carbamazepine was used as the internal standard and the sample pretreatment involved one-step protein precipitation. Chromatographic separation was conducted on an Ultimate C18 column with a mobile phase consisting of acetonitrile-water (containing 0.1% formic acid; 40:60, v/v) at a flow rate of 0.3 mL/min. The detection of voriconazole was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring with an electrospray ionization source in the positive mode. The standard curve was linear (r(2) ≥ 0.99) within the concentration range of 2.49-293 ng/mL. The intra-day and inter-day precision values were below 5.3%, and the accuracy was within -4.3-5.7%. The method was applicable to the clinical study of the pharmacokinetics of voriconazole in healthy volunteers following oral administration.

Development and validation of LC–MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine

A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine.Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200μL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer.The quantifiable range for bendamustine, M3 and M4 was 0.5–500ng/mL in plasma and 0.5–50μg/mL in urine, and that for HP2 was 1–500ng/mL in plasma and 0.1–50μg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ±20% of nominal and CV values below 20% at the lower limit of quantification and within ±15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.

A Sensitive HPLC-MS/MS Analysis of Dencichine in Rat Plasma and Its Application to Pharmacokinetics

In order to quantitate dencichine in biological samples, a selective and sensitive method for the determination of dencichine in rat plasma based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. (l)-2-amino-3-(carboxymethylthio)propionic acid was used as the internal standard (I.S.). After a protein precipitation extraction with acetonitrile, dencichine and the I.S. were chromatographed on an Xterra MS-C18 column. The mobile phase was consisted of 20mmol/L ammonium acetate solution-acetonitrile (35:65, V/V) at a flow rate of 0.2 mL/min. The detection was performed on a triple quadrupole mass via electrospray ionization (ESI) source in the positive mode. The intra- and inter-day precision (relative standard deviation, R.S.D.) values of dencichine were below 6.7%. The extraction recoveries were up 85%. The lower limit of quantification was 20 ng/ml, which was sensitive enough to detect the analyte. The HPLC-MS/MS method was successfully applied to the pharmacokinetic study after an intravenous administration of dencichine in Sprague-Dawley (SD) rat.

A Brother's Grimm true story: the valiant little triple-quadrupole - 7 allergens in one blow

The development of a multi-method for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction mode is presented. It is based on extraction of the allergenic proteins from a food matrix, followed by enzymatic digestion with trypsin. The chosen marker peptides were implemented into one method that is capable of the simultaneous detection of milk, egg, soy, hazelnut, peanut, walnut and almond. This method has been used to detect all seven allergenic commodities from incurred reference bread material, which was baked according to a standard recipe from the baking industry. The presentation demonstrates the high potential of LC-MS/MS for allergens screening for the food industry.

MS/MS studies on the selective on-line detection of sesquiterpenes using a Flowing Afterglow–Tandem Mass Spectrometer (FA-TMS)

Abstract. A Flowing Afterglow-Tandem Mass Spectrometer (FA-TMS) was used to investigate the feasibility of selective on-line detection of a series of seven sesquiterpenes (SQTs). These SQTs were chemically ionized by either H3O+ or NO+ reagent ions in the FA, resulting among others in protonated SQT and SQT molecular ions, respectively. These and other Chemical Ionization (CI) product ions were subsequently subjected to dissociation by collisions with Ar atoms in the collision cell of the tandem mass spectrometer. The fragmentation spectra show similarities with mass spectra obtained for these compounds with other instruments such as a Proton Transfer Reaction-Linear Ion Trap (PTR-LIT), a Proton Transfer Reaction-Mass Spectrometer (PTR-MS), a Triple Quadrupole-Mass Spectrometer (QqQ-MS) and a Selected Ion Flow Tube-Mass Spectrometer (SIFT-MS). Fragmentation of protonated SQT is characterized by fragment ions at the same masses but with different intensities for the individual SQT. Distinction of SQTs is based on well-chosen intensity ratios and collision energies. The fragmentation patterns of SQT molecular ions show specific fragment ion tracers at m/z 119, m/z162, m/z 137 and m/z 131 for α-cedrene, δ-neoclovene, isolongifolene and α-humulene, respectively. Consequently, chemical ionization of SQT by NO+, followed by MS/MS of SQT+ seems to open a way for selective quantification of SQTs in mixtures.

Androgen-regulated metabolome in prostate cancer.

25 Background: Prostate cancer (PC) is the second most prevalent cancer among American men which is primarily treated by androgen ablation therapy. Although a number of patients respond to this regimen, a significant subset fail and the tumor invariably progresses into a hormone refractory metastatic state, which is lethal. Earlier we had reported the first unbiased metabolomic signature for localized and metastatic prostate cancer tissues. Advancing further, we attempt to delineate the subset of metabolome in prostate cancer which is regulated by androgen-action. Methods: Androgen responsive (R22V1, LnCap and VCAP) and independent (PC3, DU145) PC cells and benign prostate epithelial cells (RWPE) were profiled for their metabolomic alterations using mass spectrometry. Extracted metabolome from these cells were profiles using a combination quadrupole-time-of-flight (Q-TOF) and triple quadrupole (QQQ) mass spectrometers coupled to reverse phase and aqueous normal phase chromatography. The metabolomic profiles were analyzed to delineate class-specific signatures which were interrogated for altered bioprocesses using Oncomine Concept Map (OCM, www.oncomine.org ). The androgen receptor (AR) regulated metabolome was verified using treatment of PC cells with synthetic androgen, R1881. Results: A total of 3,092 metabolites (113 named) were detected across the 4 cells lines, of which 869 compounds were significantly (ANOVA P<0.01) different between androgen responsive and non-responsive cells. The differential compendia included 28 named metabolites, including sarcosine which was earlier shown to be elevated during PC development and progression. Bioprocess mapping of AR-regulated metabolome revealed enrichment of amino acid metabolism and methylation potential, both of which were earlier defined to be the hallmarks of PC development and progression. Conclusions: The study defines AR-regulated metabolic signature which portrays enrichment of amino acid metabolism and methylation potential that are hallmarks of PC development and progression. No significant financial relationships to disclose.

Determination of Mildronate in Human Plasma and Urine by UPLC–Positive Ion Electrospray Tandem Mass Spectrometry

A simple, rapid, and selective method to determine the concentration of mildronate in human plasma and urine using ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS-MS) was developed and validated. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization at m/z 147.2–58.0 for mildronate and m/z 147.2–87.8 for the internal standard, carbachol. The UPLC separation was carried out with a UPLC BEH HILIC column. The mobile phase consisted of 0.08% formic acid in 30 mM ammonium acetate solution and acetonitrile (23:77, v/v). Plasma samples were extracted from plasma by protein precipitation and urine samples were diluted with the mobile phase. The analysis time was 3.5 min for each sample. Linear calibration curves ranged from 0.10 to 100.00 μg mL−1 in human plasma and 0.50 to 600.00 μg mL−1 in urine. The method had been successfully applied to a pharmacokinetic study in healthy volunteers. After single intravenously administration of 250, 500, and 750 mg mildronate, the elimination half-life (t1/2) were (2.74 ± 0.67), (4.86 ± 0.82) and (5.16 ± 0.77) h, respectively. The t1/2 for the 250 mg dose did vary significantly with other dosages (P < 0.05), mildronate may have non-linear pharmacokinetics in humans.

An LC–MS–MS Method for Quantification of CMDCK: A Novel Anti-HIV Agent in Rat Plasma and Its Application to a Pharmacokinetic Study

A simple and sensitive LC–MS–MS method was developed and validated to quantify CMDCK, a novel non-nucleoside reverse transcriptase inhibitor in rat plasma. A simple protein precipitation with acetonitrile was used to pretreat plasma samples. Chromatographic separation was carried out on a Hypersil GOLD-C18 (50 mm × 2.1 mm, 5 μm) column with the mobile phase consisting of acetonitrile and 0.1% formic acid solution (60:40 v/v) at a flow rate of 0.2 mL min−1. A triple-quadrupole tandem mass spectrometer with ESI source was operated in the positive ion mode. Quantitative analysis was conducted in the selected reaction monitoring mode with precursor/product ion transitions of m/z 698/500 and 693/495, respectively for CMDCK and IS. The calibration curves were linear over the concentration range of 5–2,000 ng mL−1 with the lower limit of quantification of 5 ng mL−1. The intra- and inter-day precisions and accuracies were satisfactory, with the coefficient of variation and the relative error being less than 13.2 and 3.38%, respectively. The recovery of CMDCK in plasma ranged from 84.6 ± 6.5 to 100.8 ± 9.3%. This LC–MS–MS method was successfully used as a new approach for the preclinical pharmacokinetics study of CMDCK in rat.

Abstract A52: Metabolomic profiling reveals impaired xenobiotic metabolism in bladder cancer

Abstract Introduction: Bladder cancer (BCa) is the second most prevalent urological malignancy and the fourth highest cause of cancer-related death in the United States. Earlier studies have linked BCa development to alterations in metabolic pathways. Significant among these are decreased activity of N-acetyl transferases causing a slow-acetylator phenotype leading to inefficient detoxification of aromatic hydrocarbons causal to onset of BCa. Interestingly, Afro-American patients inherently exhibit such slow acetylator phenotype and are known to have a more aggressive form of the tumor compared to Caucasians. This indicates existence of a metabolic niche that governs the racial disparity in BCa, which is not well understood. Also, there is an imminent need to develop non-invasive markers for early detection and prognosis of BCa, since urine cytology which is the current clinical standard is not specific to the tumor. Using mass spectrometry we report metabolic alterations in BCa and delineate bioprocesses that are altered during its progression. Our data for the first-time demonstrates role of methylation in attenuation of xenotbiotic metabolism in BCa. Furthermore, the metabolic profiles seed further analysis to examine the racial disparity in these tumors. Methods: Total metabolome from flash frozen clinically annotated bladder-derived tissues (n=58,31 benign adjacent and 27 BCa, 26 matched pairs) were examined using a combination of Q-TOF (unbiased) and triple-quadrupole (targeted) mass spectrometry. Panel of well-defined standards were used to ensure reproducibility of the profiling process. The metabolites were pre-fractionated using liquid chromatography prior to mass spectrometry in both the positive and negative ionization mode. The unbiased mass spectral data was searched using Metlin library to identify the compounds. The metabolomic profiles thus generated were analyzed to delineate class-specific signatures which were interrogated for altered bioprocesses using Molecular Concept Map (OCM, www.oncomine.org). The altered bioprocesses were validated in cell line models using a combination of Q-PCR, immunoblot analysis and functional assays. Results and discussion: A total of 2019 compounds were detected across the 58 bladder-derived specimens of which, 423 compounds were significantly altered in BCa compared to adjacent benign. 50 of the differential compounds were named and used for developing a classificatory signature and bioprocess mapping. Included among these were polycylic compounds like aniline, catechols, aromatic amino acids, polyamines and S-adenosyl methionine (SAM). Interestingly this BCa-specific metabolic signature in tissues was able to delineate tumor from benign with an accuracy of 75 %. Importantly the functional mapping of the metabolic data revealed enhanced methylation potential in tumors as being one of the factors de-regulating the xenobiotic metabolism. In vitro experiments using bisulfite sequencing and methyltransferase inhibitor 5-Aza-cytidine confirmed this methylation-induced attenuation of phase I/II metabolic genes namely CYP1A1, CYP1B1, EPHX1 and GSTT1 in BCa. In summary, using unbiased metabolomic profiling report metabolic fingerprint for bladder cancer. Importantly our data for the first time reveals methylation-induced silencing of xenobiotic metabolism in bladder tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A52.

Analysis of Nanafrocin in Foodstuffs of Animal Origin by LC–MS–MS

A new, rapid, and efficient method, multiple reaction monitoring liquid chromatography–tandem mass spectrometry, has been developed for analysis of nanafrocin in foodstuffs of animal origin. The researchers used a C18 stationary phase coupled with triple-quadrupole tandem mass spectrometry in negative-electrospray mode. The limits of detection (LOD) and quantification (LOQ) were 0.005 and 0.01 mg kg−1, respectively, in the matrixes. Detector response was found to be a linear function of concentration over the range 0.005–0.1 mg kg−1 in each matrix. Mean overall recovery (n = 10) of nanafrocin varied from 71 to 101%. The results show that identification and quantification of nanafrocin residues in foodstuffs of animal origin can be successfully achieved by use of the proposed LC–MS–MS method.

Sensitive and Selective LC–ESI–MS Analysis of Aesculin in Rat Plasma

A rapid and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the determination of aesculin in rat plasma. The analyses were chromatographed on a Zorbax Extend-C18 analytical column (150 × 2.1 mm I.D., 5 µm) with 30:70 (v/v) methanol–0.1% formic acid as mobile phase. Detection was performed by triple-quadrupole tandem mass spectrometry in multi-reaction-monitoring mode with an electrospray ionization source. The method was validated for accuracy and precision, and linearity in the two matrices was good. The assay was linear in the range 12.5–1,800 ng mL−1. The lower limit of quantification of aesculin (LLOQ) was 12.5 ng mL−1. The recovery of aesculin and tinidazole (IS) were well above 85%. The within- and between-batch accuracy was 100–104% and 97–109%, respectively. There were no stability-related problems in the procedure for the analysis of aesculin. The method was successfully used in a preclinical study of the pharmacokinetics of aesculin in rats.

Effect of Nucleoside and Nucleotide Reverse Transcriptase Inhibitors of HIV on Endogenous Nucleotide Pools

Alterations in endogenous nucleotide pools as a result of HIV therapy with nucleoside and nucleotide reverse transcriptase inhibitors (N[t]RTIs) is a proposed mechanism for therapy-related adverse events and drug interactions resulting in treatment failure. In vitro studies were performed in order to understand the effect of N(t)RTIs on endogenous nucleotide pools. The T-cell line CEM-CCRF was treated with control antimetabolites or the N(t)RTIs abacavir, didanosine, lamivudine, tenofovir (TFV) and zidovudine (AZT), either alone or in combination. The levels of natural 2'-deoxynucleoside triphosphates (dNTP) and ribonucleoside triphophosphates were determined by liquid chromatography coupled with triple quadrupole mass spectrometry. Antimetabolites altered nucleotide pools in a manner consistent with their known mechanisms of action. AZT was the only N(t)RTI that significantly altered dNTP pools. incubation of 10 microM AZT, either alone or in combination with other N(t)RTIs, increased 2'-deoxyadenosine triphosphate, 2'-deoxyguanosine triphosphate and thymidine triphosphate levels by up to 1.44-fold the concentrations observed in untreated cells. At higher than pharmacological concentrations of AZT, evidence for inhibition of 2'-deoxycytidylate deaminase and enzymes involved in the salvage of thymidine was also observed. Phosphorylated metabolites of TFV are known to inhibit purine nucleoside phosphorylase (PNP). However, in contrast to a potent PNP inhibitor, TFV was unable to alter intracellular dNTP pools upon addition of exogenous 2'-deoxyguanosine. N(t)RTIs have the potential to alter nucleotide pools; however, at the pharmacologically relevant concentrations, tested N(t)RTI or their combinations did not have an effect on nucleotide pools with the notable exception of AZT.

Simultaneous LC–MS–MS Analysis of Danshensu, Salvianolic Acid B, and Hydroxysafflor Yellow A in Beagle Dog Plasma, and Application of the Method to a Pharmacokinetic Study of Danhong Lyophilized Powder for Injection

A reliable and sensitive liquid chromatographic–tandem mass spectrometric method, with rutin as internal standard, has been developed and validated for simultaneous determination of danshensu, salvianolic acid B (SAB), and hydroxysafflor yellow A (HSYA) in beagle dog plasma. Plasma samples spiked with the analytes were extracted by solid-phase extraction and the analytes were separated on a 250 × 4.6 mm i.d., 5-μm particle, C18 column with methanol–acetonitrile–0.5% formic acid 20:25:55 (v/v) as mobile phase at a flow rate of 1 mL min−1. LC–MS–MS analysis was performed with a Finnigan TSQ triple-quadrupole tandem mass spectrometer operated in negative-ion selected-reaction-monitoring mode, using electrospray ionization. The accuracy and precision of the method were acceptable and linearity was good over the range 20–4,000 ng mL−1 for danshensu, 50–10,000 ng mL−1 for SAB, and 10–2,000 ng mL−1 for HSYA. The method was successfully applied to a pharmacokinetic study of a traditional Chinese medicinal preparation, Danhong lyophilized powder for injection.

Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.