[Comparison of gemstone spectral curve and CT value of gastric cancer with different pathological types and differentiation degrees].

Objective: To investigate the differences of gemstone spectral curve and CT value of gastric cancer with different pathological types and differentiation degrees. Methods: 91 cases of preoperative gemstone CT images with gastric cancer were collected, including 24 cases of mucinous carcinoma, 67 cases of non-mucinous carcinoma, 16 cases of signet ring cell carcinoma, 8 cases of mucinous adenocarcinoma, 32 cases of moderately differentiated adenocarcinoma and 35 cases of poorly differentiated adenocarcinoma. Gemstone CT spectral imaging was performed preoperatively, and the spectral curve of the lesion in venous phase was obtained by using GSI Viewer software, the slope of the curve was calculated, and 11 monoenergetic CT values of 40~140 keV (10 keV interval) were measured. The gemstone spectral curves and CT values of gastric cancer with different pathological types and differentiation degrees are compared. Results: The curve slopes of non-mucinous carcinoma, signet ring cell carcinoma and poorly differentiated adenocarcinoma were -1.92±0.53, -1.73±0.37 and -2.14±0.54, respectively. The absolute values were higher than those of mucinous carcinoma (-1.45±0.54), mucinous adenocarcinoma (-0.90±0.34) and moderately differentiated adenocarcinoma (-1.67±0.41), and the differences were all statistically significant (P<0.05). There were significant differences in monoenergetic CT values between mucinous and non-mucinous carcinomas at 40-140 keV (all P<0.05). The former was lower than the latter in different degrees, and the lower the energy, the greater the difference was. There were significant differences in monoenergetic CT values between signet ring cell carcinoma and mucinous adenocarcinoma at 40-100 keV (all P<0.05); monoenergetic CT values between poorly differentiated adenocarcinoma and moderately differentiated adenocarcinoma at 40-90 keV showed statistically significant differences (P<0.05). Conclusions: Gastric cancer with different pathological types and differentiation degrees have their characteristic spectral curves in venous phase, and the monoenergetic CT values are significantly different at low energy. The spectral curve of gemstone CT may be helpful to evaluate the pathological type and differentiation degree of gastric cancer before operation.

[Efficacy and safety study of microsurgical varicocelectomy in the treatment of non-obstructive azoospermia with varicocele].

Objective: To discuss the efficacy and safety of subinguinal microsurgical varicocelectomy in the treatment of non-obstructive azoospermia (NOA) with varicocele. Methods: The clinical data of 141 patients with NOA and varicocele who underwent subinguinal microsurgical varicocelectomy from March 2015 to June 2017 in Shanghai General Hospital was collected.One hundred and ten patients suffered from varicocele on the left side, 1 on the right side, and the rest (30 cases) were bilateral varicocele. Grade Ⅰ varicocele were found on 7 sides (the right and left side was count respectively), grade Ⅱ on 121 sides, and grade Ⅲ on 43 sides. Sperm analysis, pregnancy rate and complications were recorded after at least 6 months since operation. Results: Eleven cases were lost during the follow-up. Eighteen of the remaining 130 NOA patients processed successful sperm retrieval in post-operative semen analysis (18/130, 13.8%). Six couples(6/130, 4.6%) succeeded in natural pregnancy. Five couples (5/130, 3.8%)underwent successful pregnancy following with intracytoplasmic sperm injection(ICSI). Twenty-six out of the remaining 112 patients underwent the micro dissection testicular sperm extraction (micro-TESE), and 4 patients got a successful sperm retrieval (4/26, 15.4%). Among them, 2 couples had successful pregnancy with ICSI. Totally 2 cases of postoperative infection of incision were found. Conclusions: Microsurgical varicocelectomy had a beneficial effect on sperm quality of patients suffered from NOA with varicocele to some extent, even leading to unassisted pregnancy or avoiding micro-TESE before ICSI. Microsurgical varicocelectomy could be applied in the treatment of NOA with varicocele.

An integrated bioanalytical method development and validation approach: case studies.

We proposed an integrated bioanalytical method development and validation approach: (1) method screening based on analyte's physicochemical properties and metabolism information to determine the most appropriate extraction/analysis conditions; (2) preliminary stability evaluation using both quality control and incurred samples to establish sample collection, storage and processing conditions; (3) mock validation to examine method accuracy and precision and incurred sample reproducibility; and (4) method validation to confirm the results obtained during method development. This integrated approach was applied to the determination of compound I in rat plasma and compound II in rat and dog plasma. The effectiveness of the approach was demonstrated by the superior quality of three method validations: (1) a zero run failure rate; (2) >93% of quality control results within 10% of nominal values; and (3) 99% incurred sample within 9.2% of the original values. In addition, rat and dog plasma methods for compound II were successfully applied to analyze more than 900 plasma samples obtained from Investigational New Drug (IND) toxicology studies in rats and dogs with near perfect results: (1) a zero run failure rate; (2) excellent accuracy and precision for standards and quality controls; and (3) 98% incurred samples within 15% of the original values.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Effect of cinnamaldehyde on biofilm formation and sarA expression by methicillin-resistant Staphylococcus aureus.

AIMS: To investigate the antibiofilm effect of cinnamaldehyde on methicillin-resistant Staphylococcus aureus (MRSA) and analyse the effect of subminimum inhibitory concentrations (MICs) of cinnamaldehyde on the expression of the biofilm-related gene sarA. METHODS AND RESULTS: The MICs and minimum bactericidal concentrations (MBCs) were determined using a microtitre broth dilution method. Biofilm susceptibility was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining and colony forming unit (CFU) counting assays. Antibiofilm effects were studied with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). SarA expression was assessed by real-time PCR. MICs and MBCs were in the range 0.0625-0.5% (v/v). The killing effects were concentration dependent. At a concentration of 5× MIC, all strains in biofilm were decreased to lower than 20% of the control groups. SEM and CLSM images indicated that a 5× MIC concentration of cinnamaldehyde was able to detach and kill existing biofilms. Apart from strain JB-06, real-time PCR showed that the expression of sarA of all other strains was decreased upon exposure to sub-MICs of cinnamaldehyde. CONCLUSIONS: These data showed the strong killing effect of cinnamaldehyde against MRSA within biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated the potential of cinnamaldehyde as an inhibitory agent for use in MRSA biofilm-related infections.

KLF7 promotes macrophage activation by activating the NF-κB signaling pathway in epicardial adipose tissue in patients with coronary artery disease.

OBJECTIVE: Inflammatory accumulation in epicardial adipose tissue (EAT) may influence the formation and development of coronary artery disease (CAD). EAT macrophages exhibit M1 polarization and the secretion of a large number of inflammatory factors in CAD patients. Emerging data demonstrate that Krüppel-like factor-7 (KLF7), contributes to the regulation of adipocyte differentiation and the secretion of adipose tissue inflammation. However, the function of KLF7 in EAT inflammation still remains to be uncovered. This study aims to investigate the role of KLF7 in macrophage activation in EAT. PATIENTS AND METHODS: The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels were measured by Real Time-PCR. The protein expression level was detected by Western blot. RESULTS: The expression of inflammatory factors and KLF7 were markedly increased in CAD EAT than non-CAD EAT. KLF7 is highly expressed in human THP-1-derived macrophages induced by inflammatory stimuli, such as LPS. The knockdown of KLF7 inhibited the release of inflammatory factors and significantly decreased the expression of KLF7 in human THP-1-derived macrophages stimulated by LPS. Moreover, transfection with KLF7-siRNA caused the marked inhibition of LPS-induced phosphorylation of JNK-MAPKs and also suppressed the levels of p-p65 and inhibited the activation of p-IκBα. CONCLUSIONS: Taken together, these results indicate that KLF7 enhances macrophage activation, mediated by JNK-NF-κB signaling pathways in EAT. This suggests that KLF7 may be a potential therapeutic target for cardiovascular diseases such as CAD.

Preliminary study of hemodynamic distribution in patient-specific stenotic carotid bifurcation by image-based computational fluid dynamics.

BACKGROUND: Regions prone to atherosclerosis, such as bends and bifurcations, tend to exhibit a certain degree of non-planarity or curvature, and these geometric features are known to strongly influence local flow patterns. Recently, computational fluid dynamics (CFD) has been used as a means of enhancing understanding of the mechanisms involved in atherosclerotic plaque formation and development. PURPOSE: To analyze flow patterns and hemodynamic distribution in stenotic carotid bifurcation in vivo by combining CFD with magnetic resonance angiography (MRA). MATERIAL AND METHODS: Twenty-one patients with carotid atherosclerosis proved by digital subtraction angiography (DSA) and/or Doppler ultrasound underwent contrast-enhanced MR angiography of the carotid bifurcation by a 3.0T MR scanner. Hemodynamic variables and flow patterns of the carotid bifurcation were calculated and visualized by combining vascular imaging postprocessing with CFD. RESULTS: In mild stenotic cases, there was much more streamlined flow in the bulbs, with reduced or disappeared areas of weakly turbulent flow. Also, the corresponding areas of low wall shear stress (WSS) were reduced or even disappeared. As the extent of stenosis increased, stronger blood jets formed at the portion of narrowing, and more prominent eddy flows and slow back flows were noted in the lee of the stenosis. Regions of elevated WSS were predicted at the portion of stenosis and in the path of the downstream jet. Areas of low WSS were predicted on the leeward side of the stenosis, corresponding with the location of slowly turbulent flows. CONCLUSION: CFD combined with MRA can simulate flow patterns and calculate hemodynamic variables in stenotic carotid bifurcations as well as normal ones. It provides a new method to investigate the relationship of vascular geometry and flow condition with atherosclerotic pathological changes.

[Prognostic factors of pediatric patients with Ph-positive acute lymphoblastic leukemia].

Objective: To explore the clinical features and prognostic factors of Ph-positive and/or BCR-ABL positive acute lymphoblastic leukemia (Ph+ ALL) in children. Methods: The clinical data of 68 Ph+ ALL children who were treated at Peking University People's Hospital from December 2006 to December 2016 was retrospectively reviewed. Survival analysis were estimated by Kaplan-Meier method. Univariate analysis was estimated by Log-rank test and Chi-square, and multivariate analysis was estimated by Cox proportional hazards regression model. Results: In the 68 cases, the proportion of male to female was 2.1∶1, with a median age of 8 (1-16) years, and the median overall survival (OS) and disease free survival (DFS) were 16.8 months and 13.5 months, respectively. The early response rate to treatment was 43.9%, with myeloid-antigens-expression group lower than the non-expression group (29.6% vs 61.3%, χ2=5.814, P=0.020); The complete remission (CR) rate after one-course induction therapy was 86.2% (56/65), with good-response group higher than the poor-response group (100.0% vs 74.2%, χ2=6.680, P=0.003);The CR rate after induction in patients receiving imatinib plus chemotherapy was higher than the patients receiving chemotherapy only (94.9% vs 73.1%, χ2=5.185, P=0.024). The 2-and 5-year OS were (61.4±7.0)% and (50.8±8.1)%, respectively. The 2-and 5-year DFS were (54.6±6.8)% and (48.6±7.3)%, respectively. Univariate analysis showed that the initial WBC, LDH, spleen size, liver size, with-myeloid-antigens-expression, early response to treatment, MRD (BCR-ABL) after one-course induction, application of imatinib and different treatment options affected 2-year OS rate (all P<0.05). LDH, spleen size, liver size, with-myeloid-antigens-expression, early response to treatment, MRD (BCR-ABL) after one-course induction, application of imatinib and different treatment options affected 2-year DFS rate (all P<0.05). Multivariate prognostic analysis for OS (RR=45.7, 95% CI 1.4-1 528.2, P=0.033) and DFS (RR=52.3, 95% CI 1.6-1 725.9, P=0.026) showed that the spleen ≥ 3 cm was the independent risk factor. Conclusions: Pediatric Ph+ ALL is a special condition with unique clinical and biological features. The early response to treatment was poor in patients with myeloid-antigens-expression, which resulted in a low CR rate after one-course induction and the administration of imatinib can remarkably improve the CR rate. Initial spleen ≥ 3 cm is an independent prognostic factor. The efficacy of chemotherapy alone is poor, and imatinib combined with chemotherapy is applauded in the aim of improving outcomes.

Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry.

BMS-299897 is a gamma-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid-liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized (13)C6-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm x 50 mm, 3 microm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25-100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within +/-7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.

Dual PPARα/γ ligand TZD18 improves myocardial metabolic remodeling after myocardial infarction in rats.

OBJECTIVE: To investigate the changes in myocardial energy metabolism and the effect of peroxisome proliferator-activated receptor alpha/gamma (PPARα/γ) dual agonist TZD18 on myocardial energy metabolism in rats with heart failure after myocardial infarction. MATERIALS AND METHODS: The myocardial infarction model was established by ligating the left anterior descending coronary artery. The rats were randomly divided into the myocardial infarction group (MI group), the TZD18 intervention group (TZD18 group), and the shame surgery group (sham group). 8 weeks later, the blood flow parameters were measured by carotid arterial cannulas, and ventricular remodeling indexes were calculated. Hearts were extracted from rats after the execution. The expressions of PPARα/γ mRNA and α/ß-MHC mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). The mitochondrial oxidative respiration activity was measured by a bio-tissue oxygen consumption meter, the content of adenosine in mitochondria was measured by high-performance liquid chromatography, and tritium-labeled adenosine diphosphate incorporation assay was used to detect the transport activity of adenosine nucleotide translocases (ANT). RESULTS: The expression of PPARα/γ mRNA and the ratio of α/ß-MHC mRNA in the MI group were significantly decreased, the content of high energy phosphates, respiration activity, ANT transport activity in mitochondria were significantly decreased, the hemodynamic indexes were disturbed and left ventricular weight/body weight ratio (LVW/BW) significantly became higher. TZD18 intervention could increase the expression level of PPARα/γ mRNA and up-regulate the ratio of α/ß-MHC mRNA, thus improving mitochondrial respiratory activity and ANT transport activity in rats with heart failure after myocardial infarction, increasing the content of high energy phosphates in mitochondria and improving the remodeling indexes in the ventricle. CONCLUSIONS: TZD18 increases both the expression of enzymes related to myocardial energy metabolism and the content of high-energy phosphates in mitochondria. Also, it improves the respiratory activity and ANT transport activity by activating PPARα/γ genes, thus improving the generation and delivery of myocardial energy and protecting the myocardial cells.

Integrative analyses of transcriptome sequencing identify novel functional lncRNAs in esophageal squamous cell carcinoma.

Long non-coding RNAs (lncRNAs) have a critical role in cancer initiation and progression, and thus may mediate oncogenic or tumor suppressing effects, as well as be a new class of cancer therapeutic targets. We performed high-throughput sequencing of RNA (RNA-seq) to investigate the expression level of lncRNAs and protein-coding genes in 30 esophageal samples, comprised of 15 esophageal squamous cell carcinoma (ESCC) samples and their 15 paired non-tumor tissues. We further developed an integrative bioinformatics method, denoted URW-LPE, to identify key functional lncRNAs that regulate expression of downstream protein-coding genes in ESCC. A number of known onco-lncRNA and many putative novel ones were effectively identified by URW-LPE. Importantly, we identified lncRNA625 as a novel regulator of ESCC cell proliferation, invasion and migration. ESCC patients with high lncRNA625 expression had significantly shorter survival time than those with low expression. LncRNA625 also showed specific prognostic value for patients with metastatic ESCC. Finally, we identified E1A-binding protein p300 (EP300) as a downstream executor of lncRNA625-induced transcriptional responses. These findings establish a catalog of novel cancer-associated functional lncRNAs, which will promote our understanding of lncRNA-mediated regulation in this malignancy.

2017 White Paper: rise of hybrid LBA/LCMS immunogenicity assays (Part 2: hybrid LBA/LCMS biotherapeutics, biomarkers & immunogenicity assays and regulatory agencies' inputs).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for biotherapeutics, biomarkers and immunogenicity assays using hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (LCMS for small molecules, peptides and small molecule biomarkers) and Part 3 (LBA: immunogenicity, biomarkers and pharmacokinetic assays) are published in Volume 9 of Bioanalysis, issues 22 and 24 (2017), respectively.

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of muraglitazar, a novel diabetes drug, in human plasma.

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of muraglitazar, a PPAR alpha/gamma dual agonist, in human plasma. The internal standard, a chemical analogue, was dissolved in acetonitrile containing 0.1% formic acid. The solvent system was also served as a protein precipitation reagent. Human plasma samples (0.1 mL) and the internal standard solution (0.3 mL) were added to a 96-well plate. The plate was vortexed for 1 min and centrifuged for 5 min. Then the supernatant layers were directly injected into the LC/MS/MS system. The chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 20/80 (v/v) of water and acetonitrile containing 0.1% formic acid. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 1 to 1000 ng/mL, was fitted to a 1/x weighted quadratic regression model. This single-pot approach effectively eliminated three time consuming sample preparation steps: sample transfer, dry-down, and reconstitution before the injection, while it preserved all the benefits of the traditional protein precipitation. By properly adjusting the autosampler needle offset level, only the supernatant was injected, without disturbing the precipitated proteins in the bottom. As a result, the quality of chromatography and column life were not compromised. After more than 600 injections, there was only slightly increase of column back-pressure. The validation results demonstrated that this method was rugged and provide satisfactory precision and accuracy. The method has been successfully applied to analyze human plasma samples in support of a first-in-man study. This method has also been validated in monkey and mouse plasma for the determination of muraglitazar.

A 96-well single-pot liquid-liquid extraction, hydrophilic interaction liquid chromatography-mass spectrometry method for the determination of muraglitazar in human plasma.

A single-pot liquid-liquid extraction (LLE) with hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC/MS/MS) method has been developed and validated for the determination of muraglitazar, a hydrophobic diabetes drug, in human plasma. To 0.050 ml of each plasma sample in a 96-well plate, the internal standard solution in acetonitrile and toluene were added to extract the compound of interest. The plate was vortexed, followed by centrifugation. The organic layer was then directly injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Thermohypersil_Keystone, Hypersil silica column (3 mmx50 mm, 3 microm). The mobile phase contained 85% of methyl t-butyl ether and 15% of 90/10 (v/v) acetonitrile/water with 0.3% trifluoroacetic acid. Post-column mobile phase of 50/50 (v/v) acetonitrile/water containing 0.1% formic acid was added. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 4000. The standard curve, ranged from 1 to 1000 ng/ml, was fitted to a 1/x weighted quadratic regression model. This single-pot LLE approach effectively eliminated time-consuming organic layer transfer, dry-down, and sample reconstitution steps, which are essential for a conventional liquid-liquid extraction procedure. The modified mobile phase was more compatible with the direct injection of the commonly used extraction solvents in LLE. Furthermore, the modified mobile phase improved the retention of muraglitazar, a hydrophobic compound, on the normal phase silica column. The validation results demonstrated that this method was rugged and suitable for analyzing muraglitazar in human plasma. In comparison with a revised-phase LC/MS/MS method, this single-pot LLE, HILIC/MS/MS method improved the detection sensitivity by more than four-fold based upon the LLOQ signal to noise ratio. This approach may be applied to other hydrophobic compounds with proper modification of the mobile phase compositions.

Use of 4ß-hydroxycholesterol in animal and human plasma samples as a biomarker for CYP3A induction.

BACKGROUND: 4ß-hydroxycholesterol (4ßHC) has recently been proposed as a potential endogenous biomarker for CYP3A activity. Developing bioanalytical assays for 4ßHC is challenging for several reasons, including endogenous background levels in plasma; the presence of free and ester forms; the inherent lack of MS sensitivity; and the presence of multiple positional isomers. RESULTS: Bioanalytical assays in mouse, rat, dog and human plasma were adapted and modified from a previous published human plasma assay for 4ßHC by using alkaline de-esterification, picolinic derivatization, a surrogate analyte (d7-4ßHC) in authentic matrices and chromatographic conditions that showed good separation from isobaric, positional isomers. CONCLUSION: These assays were applied to multiple studies and demonstrated potential applications of 4ßHC as a CYP3A biomarker across preclinical and clinical settings.

Quantitative determination of pioglitazone in human serum by direct-injection high-performance liquid chromatography mass spectrometry and its application to a bioequivalence study.

A simple, high throughput, direct-injection high-performance liquid chromatography tandem mass spectrometry method (LC/MS/MS) has been developed and validated for the quantitation of pioglitazone in human serum. After mixing the internal standard with a sample, a 10 microl portion of the mixture was directly injected into a high-flow LC/MS/MS system, which included an extraction column, an analytical column and a six-port switching valve. The on-line extraction was achieved on an Oasis HLB column (1 mm x 50 mm, 30 microm) with a 100% aqueous loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a flow rate of 4 ml/min. The extracted analyte was eluted by a mobile phase which contained 5 mM ammonium acetate and acetonitrile. The analytical column was a Luna C18 column (4.6 mm x 50 mm, 5 microm). Detection was achieved by positive ion electrospray tandem mass spectrometry. The lower limit of quantitation of the method was 9 ng/ml. The standard curve, which ranged from 9 to 1350 ng/ml, was fitted by a weighted (1/x2) quadratic regression model. The validation results demonstrated that this method had satisfactory precision and accuracy across the calibration range. There was no evidence of instability of the analyte in human serum following three freeze-thaw cycles, and samples could be stored for at least 2 weeks at -30 degrees C. This method was used to analyze pioglitazone concentrations in human serum samples from a bioequivalence study of a blinded Actos formulation (encapsulated 15 mg tablet) and an Actos 15 mg tablet. The blinded formulation was shown to be bioequivalent to an Actos 15 mg tablet.

A simple 96-well liquid-liquid extraction with a mixture of acetonitrile and methyl t-butyl ether for the determination of a drug in human plasma by high-performance liquid chromatography with tandem mass spectrometry.

A simple 96-well plate liquid-liquid extraction (LLE), liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a basic drug candidate in human plasma. Against the wisdom of conventional approaches, an aqueous/organic miscible solvent, acetonitrile, was used for liquid-liquid extraction along with methyl t-butyl ether. The use of acetonitrile effectively eliminated the formation of the irregular emulsion between aqueous/organic interfaces and modulated the polarity of the extraction solvents to achieve the desired recovery. This approach, which solved the emulsion problem, permitted the method to be automated using standard 96-well plate technology. A practical application was demonstrated through the use of this technique in the measurement of a novel drug in human plasma samples by LC/MS/MS. Chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 60% of 0.1% formic acid and 40% acetonitrile. Detection was by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 1.22 to 979ng/ml, was fitted to a 1/x2 weighted quadratic regression model. The validation results show that this method was very rugged and had excellent precision and accuracy. The actual sample analysis results further demonstrated that this extraction procedure is well suited for real life applications. It is expected that with some modifications, this approach can be applied for the extraction of similar compounds from various biological fluids.

Bioanalysis of drug in tissue: current status and challenges.

Distribution of drugs into tissues is an important determinant of the overall PK and PD profile. Thus, bioanalysis of drugs and their metabolites in tissues can play an important role in understanding the pharmacological and toxicological properties of new drug candidates. Unlike liquid matrices, bioanalysis in tissues offers unique challenges such as proper tissue sampling, appropriate tissue sample preparation, efficient extraction of the analytes from the tissue homogenates, and demonstration of stability and recovery of analytes in intact tissues. This article provides a systematic review of tissue sample analysis for small molecules using LC-MS/MS. The authors provide rationale for tissue sample analysis, and discuss strategies for method development, method qualification or validation, and sample analysis. Unique aspects of method development and qualification/validation are highlighted based on authors' direct experiences and literature summary. Analysis using intact tissue samples such as MALDI imaging is also briefly discussed.