Identification of plasma protein biomarkers associated with idiopathic pulmonary arterial hypertension

We have employed SELDI-TOF MS to screen for differentially expressed proteins in plasma samples from 27 patients with idiopathic pulmonary arterial hypertension (IPAH) and 26 healthy controls. One ion (m/z approximately 8600) that was found to be elevated in IPAH was validated by SELDI-TOF MS analysis of a second and separate set of plasma samples comprising 30 IPAH patients and 19 controls. The m/z 8600 was purified from plasma by sequential ion exchange and reverse-phase chromatographies and SDS-PAGE. It was identified, following trypsin digestion, by MS peptide analysis as the complement component, complement 4a (C4a) des Arg. Plasma levels of C4a des Arg measured by ELISA confirmed that the levels were significantly higher (p < 0.0001) in IPAH patients (2.12 +/- 0.27 microg/mL) compared with normal controls (0.53 +/- 0.05 microg/mL). A cut-off level of 0.6 microg/mL correctly classified 92% of IPAH patients and 80% of controls. Further studies will be needed to determine its performance as a diagnostic biomarker, whether used alone or in combination with other biomarkers. Nevertheless, this study demonstrates that putative biomarkers characteristic of IPAH can be identified using a conjoint SELDI-TOF MS - proteomics approach.     

Limitations in quantitation of the biomarker CCL18 in Gaucher disease blood samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

SELDI-TOF MS assisted the discovery of the chemokine CCL18/PARC as plasma biomarker for pathological storage cells in Gaucher disease patients. Prognostic elevation of CCL18 in blood of Gaucher patients has been confirmed by ELISA. Given its low molecular mass, positive charge, and relatively high abundance, CCL18 seems a particular attractive protein for SELDI-TOF based quantitation. Therefore, we determined CCL18 levels in plasma using SELDI-TOF MS and ELISA, in parallel. CCL18 levels in some blood samples were significantly underestimated when determined by SELDI-TOF MS. Spiking of recombinant CCL18 indicated that its detection by SELDI-TOF MS is strongly determined by the nature of the sample, even markedly varying between samples obtained from one donor at different time points. Independent of the total CCL18 concentration in blood only 1-10% of the chemokine bound to the ProteinChip Array. Even when comparable amounts of CCL18 from distinct samples were bound to the ProteinChip Array, diverse peak intensities could be observed. Thus, limited binding capacity and sample-dependent suppression of CCL18 ionization contribute significantly to the final peak intensity. In conclusion, SELDI-TOF MS offers no reliable procedure to quantitatively monitor CCL18 levels in blood and thus cannot be applied in evaluation of disease status of Gaucher patients.     

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland. Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)5(1)[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Proteomic characterization of inter-alpha inhibitor proteins from human plasma

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.     

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.     

Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2'-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC-MS/MS

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2'-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. (15)N(5)-8-OHdG ((15)N(5)-8-hydroxy-2'-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284→168 for 8-OHdG and m/z 289→173 for (15)N(5)-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.     

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.     

Centrally administered galanin modifies vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of euhydrated and dehydrated rats.

Galanin (Gal) as a neuropeptide with widespread distribution in the central nervous system may be involved in the mechanisms of vasopressin (AVP) and oxytocin (OT) release from the hypothalamo-neurohypophysial system. Vasopressin and oxytocin content in the hypothalamus and neurohypophysis as well as plasma level of both neurohormones were studied after galanin treatment in euhydrated and dehydrated rats. In not dehydrated rats intracerebroventricular (i.c.v.) injections of Gal did not affect the hypothalamic and neurohypophysial OT content, however, distinctly increased plasma OT concentration. In the same animals Gal diminished the hypothalamic AVP content but was without the effect on neurohypophysial AVP storage; plasma AVP level then raised. Galanin, administered i.c.v. to rats deprived of water, distinctly inhibited AVP and OT release from the hypothalamo-neurohypophysial system. Simultaneously, plasma AVP and OT level was significantly diminished after Gal treatment in dehydrated rats. These results suggest that modulatory effect of galanin on vasopressin and oxytocin release depends on the actual state of water metabolism. Gal acts as an inhibitory neuromodulator of AVP and OT secretion under conditions of the dehydration but stimulates this process in the state of equilibrated water metabolism.     

A high-performance liquid chromatography-tandem mass spectrometry method for the clinical combination study of carboplatin and anti-tumor agent eribulin mesylate (E7389) in human plasma

An LC/MS/MS method was developed to quantify carboplatin and eribulin mesylate (E7389) in human plasma and urine. For carboplatin, sample clean-up by protein precipitation and supernatant injection into a Waters Spherisorb((R)) S5 SCX column was used. Liquid-phase extraction and reverse-phase chromatography on a Polaris C18 column were used for eribulin. Quantitation involved LC/MS/MS with positive electrospray ionization. Accuracy, precision, linearity, range, specificity, recovery and stability were also evaluated. Both compounds were stable in human plasma (>or=80 days at -80 degrees C), at room temperature (>or=4h), following three freeze-thaw cycles and in 50/50 methanol/H(2)O (<4 degrees C for >or=252 days).     

Determination of hydroxy metabolites of polychlorinated biphenyls in plasma and tissue by gas chromatography/mass spectrometry

This study examines a novel sample preparation method for the determination of 11 hydroxy metabolites of polychlorinated biphenyls (PCBs) in plasma and organ tissues, followed by gas chromatography with mass spectrometric detection (GC/MS). The clean-up method was optimized to eliminate the interference matter by using a silica column and 10 mL of n-hexane/dichloromethane (4:6, v/v) as an eluent. Solid-phase and solvent extraction procedures were used for the plasma and tissues samples, respectively. Compared to C(18) and C(8) solid-phase, C(2) showed higher extraction efficiency with n-hexane as the eluent for plasma. The hydroxy-PCB extraction recoveries achieved with this combined extraction and clean-up procedure from plasma ranged from 87 to 117%, while those from tissues ranged from 82 to 111%. The linear detector responses for propyl derivatives of hydroxy-PCBs were obtained with the coefficients of determination varying from 0.992 to 0.998 in the concentration range of 0.1-20 ng mL(-1). The method detection limits ranged from 0.1 to 0.5 ng mL(-1) in 1 mL of plasma and from 0.1 to 0.5 ng g(-1) in 1g of tissues. This procedure was successfully applied to the study of 3-OH-2,3',4,4',5-PeCB in rat plasma and liver samples after intraperitoneal injection (20 mg/kg) of 2,3',4,4',5-PeCB.     

Effects of prostaglandin E2 and D2 on the release of vasopressin and oxytocin

The effects of prostaglandin (PG) E2 and D2 on the plasma levels of arginine-vasopressin (AVP) and oxytocin (OXT) in rabbits, and on the release of the both hormones from the isolated posterior pituitary of rats were examined. An intraventricular administration of PGE2 to a rabbit raised the plasma levels of the both hormones. An intraventricular injection of PGD2 also increased the plasma level of OXT but not that of AVP. The release of AVP and OXT from fragments of the posterior pituitary of a rat was not influenced by perfusion with Eagle MEM medium containing 10(-6) or 10(-5) M PGE2 and D2.     

SELDI-TOF MS analysis of the Cardiac Troponin I forms present in plasma from patients with myocardial infarction

The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.     

Relevance of a combined UV and single mass spectrometry detection for the determination of tenofovir in human plasma by HPLC in therapeutic drug monitoring

A sensitive high-performance liquid chromatography method coupled to UV and single mass spectrometry (MS) detection was developed for the determination of tenofovir in human plasma. A solid phase extraction procedure (Bond-Elut C18 Varian cartridges) provided high extraction efficiency (91% for tenofovir and 68.8% for the internal standard, 3-methylcytidine). An atlantis-dC-18 analytical column is used with an isocratic mode elution of a mixture (pH 2.5) of ammonium acetate/methanol (98.5:1.5, v/v). Detection was performed at 260 nm and by using the ion at m/z 288. The signals from both detectors were validated over the range of 10-1000 ng mL(-1) and were found to be linear, accurate and precise. At the lowest limit of quantification, 10 ng mL(-1) for UV and 5 ng mL(-1) for MS, the average coefficient of variation was 6.9 and 3.9%, respectively. To investigate the potential of the validated method for clinical studies, more than 170 samples from HIV-infected adult patients were then analyzed with this assay. A good correlation was observed between the results obtained with both detectors. However, in several cases discordant results were observed between UV and MS detections. Therefore, tenofovir can sometimes suffer from interferences using either UV or single MS detection. We concluded that the double detection allows to obtain a more specific quantification of tenofovir. The present assay is sound and can be used for therapeutic drug monitoring allowing a higher reliability of the results which are transmitted to the medical team.     

Enzyme immunoassay for oxytocin

A competitive, double antibody enzyme immunoassay for oxytocin in a heterologous system was developed. Horseradish peroxidase was conjugated with oxytocin using N-succinimidyl 3-(2-pyridyldithio) propionate, and rabbit anti-oxytocin serum was produced by immunization of oxytocin-bovine serum albumin complex which was prepared by the carbodiimide method. The sensitivity of the assay was 4 microIU/tube, which corresponded to 10 microIU per ml using 400 microliters of the sample which was extracted from the same volume of plasma by means of SEP-PAK C18 cartridges. The coefficients of variation for different levels of oxytocin ranged from 6.8-15.9% and 8.5-16.7%, for intra- and inter-assay. Recovery of oxytocin added to plasma after extraction was 99-117%. No or little cross-reaction with arginine- and lysine-vasopressin was found. Plasma oxytocin concentrations determined by the proposed enzyme immunoassay were well correlated with those determined by radioimmunoassay (r = 0.90).     

Vasopressin and oxytocin release as influenced by thyrotropin-releasing hormone in euhydrated and dehydrated rats.

Since the thyrotropin-releasing hormone (TRH) can modulate the processes of vasopressin (AVP) and oxytocin (OT) biosynthesis and release mainly at the hypothalamo-neurohypophysial level, the present experiments were undertaken to estimate whether TRH, administered intravenously in different doses, modifies these mechanisms under conditions of osmotic stimulation, brought about by dehydration. AVP and OT contents in the hypothalamus and neurohypophysis as well as plasma levels of AVP, OT, free thyroxine (FT4) and free triiodothyronine (FT3) were studied after intravenously TRH treatment in euhydrated and dehydrated for two days male rats. Under conditions of equilibrated water metabolism TRH diminished significantly the hypothalamic and neurohypophysial AVP and OT content but was without the effect on plasma oxytocin level; however, TRH in a dose of 100 ng/100 g b.w. raised plasma AVP level. TRH, injected i.v. to dehydrated animals, resulted in a diminution of AVP content in the hypothalamus but did not affect the hypothalamic OT stores. After osmotic stimulation, neurohypophysial AVP and OT release was significantly restricted in TRH-treated rats. Under the same conditions, injections of TRH were followed by a significant decrease of plasma OT level. I.v. injected TRH enhanced somewhat FT3 concentration in blood plasma of euhydrated animals but diminished FT4 plasma level during dehydration. Data from the present study suggest that TRH displays different character of action on vasopressin and oxytocin secretion in relation to the actual state of water metabolism.     

Radioimmunoassay of arginine vasopressin in human plasma.

Antibodies for the radioimmunoassay of arginine vasopressin (AVP) described here were produced in rabbits using synthetic AVP coupled to rabbit gamma-globulin with carbodiimide. In three out of six rabbits, significant antibody titres were obtained. Using the best antisera produced, 40% of labeled AVP was bound at a final dilution of 1:50.000. After iodination of synthetic AVP with 125I using the chloramin-T method, a gel filtration on Sephadex G-25 was performed to purify the iodinated AVP. For separation of antibody bound and free hormone, a second antibody precipitation was used. There was no crossreactivity with oxytocin. AVP was extracted from plasma after ammoniumsulfate precipitation of the proteins by adsorption to Florisil. The recovery of AVP added to plasma in amounts between 5-25 pg/ml was 60 +/- 15% (n equals 6). The minimum amount of AVP detectable was 1 pg per ml plasma. The plasma level in normal adults under standard conditions was 3.4 +/- 2.2 pg/ml. This is in agreement with data recently published by other researchers. The applicability and reproducibility was further tested in measurements of samples taken hourly during the entire day under water diuresis and after hormonal stimulation of AVP.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Determination of salirasib (S-trans,trans-farnesylthiosalicylic acid) in human plasma using liquid chromatography-tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of salirasib (S-trans,trans-farnesylthiosalicylic acid, FTS) in human plasma. Sample pretreatment involved liquid-liquid extraction with methyl t-butyl ether of 0.5-mL aliquots of lithium heparin plasma spiked with the internal standard, S-trans,trans-5-fluoro-farnesylthiosalicylic acid (5-F-FTS). Separation was achieved on Waters X-Terra C(18) (50 mm x 2.1 mm i.d., 3.5 microm) at room temperature using isocratic elution with acetonitrile/10 mM ammonium acetate buffer mobile phase (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.20 mL/min. Detection was performed using electrospray MS/MS by monitoring the ion transitions from m/z 357.2-->153.0 (salirasib) and m/z 375.1-->138.8 (5-F-FTS). Calibration curves were linear in the concentration range of 1-1000 ng/mL. A 5000 ng/mL sample that was diluted 1:10 (v/v) with plasma was accurately quantitated. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical method (<8.0%). This assay was subsequently used for the determination of salirasib concentrations in plasma of cancer patients after oral administration of salirasib at a dose of 400 mg.     

Development and validation of a highly rapid and sensitive LC-MS/MS method for determination of SZ-685C, an investigational marine anticancer agent, in rat plasma--application to a pharmacokinetic study in rats

A sensitive and rapid method was developed and validated for the quantitative analysis of the novel anticancer agent SZ-685C in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in negative ion mode in order to support the following pre-clinical and clinical studies. SZ-685C and the internal standard (IS, emodin) were extracted from rat plasma by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. Chromatographic separation was performed on an Elite Hypersil BDS C18 column (100 mm × 2.1 mm i.d., 3 μm). Elution was carried out using methanol/acetonitrile/2mM ammonium formate (pH 4) (80:15:5 (v/v/v)) at a flow-rate of 0.3 mL/min with a run time of 2.5 min. This assay was linear over a concentration range of 50-10,000 ng/mL with a lower limit of quantification of 50 ng/mL. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 100, 1000 and 7500 ng/mL. These results indicate that the method was efficient with a short run time and acceptable accuracy, precision and sensitivity. This method was successfully applied to explore pharmacokinetics of SZ-685C in rats after oral and intravenous administration of this agent. The absolute bioavailability is about 54.8-66.8% and the t(1/2) is 5.7-9.2h, these results provide basic information for further comprehensive pre-clinical research.