Analysis Of Quality Control Samples Research Articles

Simultaneous spectrophotometric and chromatographic determination of Ketorolac and Dexamethasone: Overall assessment regarding environmental Impact, Practicality, and functionality principles

Determination of a drug combination that contains a minor component that is also a poorly UV absorbing analyte represents greet challenges in drug analysis. A fixed-dose ophthalmic combination of ketorolac tromethamine (KET) and dexamethasone sodium phosphate (DEX) (in the ratio of 5:1) is used to relieve post-surgical inflammation and seasonal allergic conjunctivitis due to their analgesic and anti-inflammatory activity. Five spectrophotometric methods and a reversed phase chromatographic separation method were developed and validated for simultaneous determination of both drugs in lab-prepared eye drops. Direct spectrophotometric determination of KET (the major, highly UV absorbing analyte) was achieved from the zero-order UV absorption spectra at 323 nm while DEX (which is the minor, poorly UV absorbing analyte) was quantified after applying several spectrophotometric methods. Either fundamental zero-order spectra or ratio spectra were used, and measurements were done at either single or dual wavelengths. The developed methods were: isosbestic point method, absorption factor method, dual wavelength method, ratio difference UV spectrophotometric method, and simultaneous equation method. The developed spectrophotometric methods determine KET in the linearity range of 2.0–12.0 μg/mL and DEX in the range of 5.0 – 35.0 μg/mL. For accurate spectrophotometric determination of DEX in the ratio of the dosage form, multiple standard addition method was applied. HPLC technique was also applied for separation and quantification of both drugs. Chromatographic separation was attained by using Hypersil ODS-C18 (150 x 4.6 mm, 5 μm) column. The mobile phase was composed of 50 % methanol: 50 % phosphate buffer (pH 2.5). The column compartment was maintained at 25 °C. Flow rate was adjusted at 1.2 mL/min and detection was performed by DAD at 254 nm. The linearity of HPLC method was 5.0–100.0 μg/mL, and 5.0–50.0 μg/mL for KET and DEX, respectively. Simultaneous determination of both drugs was applied to lab-prepared eye drops that contain also benzalkonium chloride as inactive ingredient. The developed methods determined the cited drugs with good and acceptable % recovery. The results of the developed methods and a reported HPLC method were compared using one-way ANOVA test, F-test and student’s t-test at confidence level 95 %, and they show no significant statistical difference. Assessment of greenness of the developed methods in comparison with reported HPLC method was performed using different tools, including analytical eco-scale, GAPI and AGREE. HPLC-EAT was also performed to ensure greenness of developed HPLC method. Moreover, assessment of practicality, applicability, and functionality of developed and reported methods was evaluated by BAGI metric tool and WAC calculator, respectively. The developed methods allow simple, rapid, and accurate analysis of quality control samples that contain KET and DEX.

A Novel Stability Indicating RP-HPLC with PDA Approach for Estimation of Voclosporin in Bulk and Marketed Formulation

An easy, specific, and reliable method for determining voclosporin has been established using the reversed phase-high performance liquid chromatography (RP-HPLC) approach. Chromatographic conditions included stationary phase C18 Kromasil (250 mm x 4.6 mm and 5 μm), solvent system (0.1% OPA: Acetonitrile) in a 60:40 ratio, 1-mL/min flow rate, and detection wavelength of 282 nm were opted to separate the voclosporin with retention time of 2.2 minutes. A linearity analysis was performed between 3.95 to 23.7 μg/mL, and the R2 value was found to be 0.999. Precision’s %RSD was determined to be between 0.4 and 0.7. The limit of detection (LoD) and limit of quantitation (LoQ) values are 0.01 and 0.03 μg/mL, respectively. Using the aforesaid approach, the %assay of the marketed formulation was 99.07%. To test the stability representing characteristics of the suggested approach, forced degradation experiments of voclosporin were performed and %degradation was measured. Because the procedure was easy, precise, accurate, and cost-effective, it may be poted for regular analysis of quality control samples in industry

A new HPLC methodology for the analysis of metronidazole and dexibuprofen: Application to pharmacokinetic study and comparative greenness assessment

For the simultaneous analysis of metronidazole and dexibuprofen in real human plasma samples, a new HPLC method with a 6-minute analysis time was developed. Furthermore, the method was used to investigate their pharmacokinetics and bioavailability. The internal standard was chosen to be guaifenesin (IS). Following acetonitrile precipitation of plasma proteins, the drugs were chromatographically separated using a Zorbax Eclipse® C 18 column with acetonitrile: water (pH 4 with aqueous phosphoric acid) gradient mixture. Metronidazole and dexibuprofen calibration curves were found to be linear over the ranges of 0.5–20 and 1–30 µg/mL, respectively, with good correlation coefficients. The analysis of quality control samples revealed outstanding accuracy and precision. Drug stability was also examined throughout storage and processing, and all coefficients of variation were under 8.23 %. After this optimized method was successfully applied to plasma samples and the pharmacokinetic parameters of each of the targeted medicines were examined separately and compared to their parameters when they were administered concurrently. The outcomes supported each drug's significant impact on the pharmacokinetic characteristics of the other. Using five metrics, including the Analytical Eco-scale, Analytical GREEnness Calculator (AGREE), RGB-12, Hexagon, and AGREE-prep, the environmental impact of the suggested approach and some reported methods utilized for the determination of either medicines in biological matrix were evaluated. The promising results demonstrated lower environmental and health risks associated with sample preparation and analysis.

Evaluation of dried blood spots as an alternative matrix for therapeutic drug monitoring of abiraterone and delta(4)-abiraterone in prostate cancer patients

Therapeutic drug monitoring (TDM) approaches may benefit patients treated with abiraterone acetate (AA) as drug efficacy is imprecise and important pharmacokinetic variability is known. Current methods based on the analysis of plasma present the disadvantage of the fast degradation of the analytes in the liquid sample. Dried blood spots (DBS) consist of a minimally invasive and unexplored sampling strategy to monitor the levels of abiraterone (ABI) and delta(4)-abiraterone (D4A) in patients. This study presents the development and validation of a precise and accurate method to monitor ABI and D4A in DBS samples by UPLC-MS/MS. Bioanalytical method validation was carried out according to current guidelines, evaluating the impact of DBS-specific parameters such as hematocrit and spot volume on accuracy. Based on the analysis of quality control samples prepared at low, medium and high concentrations, the method was precise with CV ≤ 6.97 % and 10.26 % for ABI and D4A, respectively. The method was also highly accurate, between 93.6–106.8 % for ABI and 96.0–108.5 % for D4A. The DBS method is compatible with the analysis of samples of unknown volume and hematocrit range of the studied population. In addition, ABI and D4A were stable for 7 days in DBS at room temperature, which is feasible for sample transportation in postal service and analysis in the laboratory. Method application to 16 clinical samples revealed good correlation between measured plasma concentrations and estimated plasma concentrations for ABI (r = 0.884, P < 0.05) and D4A (r = 0.920, P < 0.05). Passing-Bablok regression analysis and Bland-Altmann plots indicated correlation between the results obtained from DBS and plasma, with a slight overestimation of the concentrations of ABI in DBS, which could be related to the small study cohort. Therefore, the results of this first work indicate that DBS consist of a promising alternative sampling strategy in TDM studies of AA.

A quick UPLC-MS/MS method for therapeutic drug monitoring of abiraterone and delta(4)-abiraterone in human plasma.

Abiraterone acetate efficacy against prostate cancer is dependent on the circulating levels of abiraterone and its active metabolites, which present significant pharmacokinetic variability among patients. Thus, therapeutic drug monitoring can be performed to improve treatment outcomes. To support such studies, there are only a limited number of bioanalytical methods in current literature. This work presents a fast method to quantify abiraterone and D4A in plasma in 4 min by UPLC-MS/MS. Bioanalytical method validation was performed according to the recommendations of the US Food and Drug Administration. The method was linear within the range of 1-400 ng/ml for abiraterone and 0.2-20 ng/ml for D4A (r2 > 0.99). Based on the analysis of quality control samples at the lower limit of quantification, low, medium and high concentrations, the method was precise (CVabiraterone ≤ 9.72%; CVD4A ≤ 14.64%) and accurate (CVabiraterone 95.51-107.59%; CVD4A 98.04-99.89%). Application of the method to the quantification of abiraterone and D4A in 10 clinical samples revealed important variability in the conversion ratio of abiraterone to D4A (CV 90.85%). Considering the current literature, this is the fastest method to quantify abiraterone and D4A in plasma, allowing for optimization of the analytical routine.

Application of drug–metal ion interaction principle in conductometric determination of imatinib, sorafenib, gefitinib and bosutinib

AbstractAn analytical method for the quantification of anticancer agents such as imatinib, sorafenib, gefitinib and bosutinib using conductometry was developed. Each drug solution was mixed with measured concentration of metal ion (Cu2+) solution resulting in drug–metal ion complexation in the titration cell. Conductance was progressively decreased on addition of the analyte solution up to a point of maximum reduction, that is, the end point. Corrected conductance values were calculated from the observed conductance and used to plot a graph against the volume of drug solution added. No interferences were observed from blank and placebo as they gave no clear inflection in the conductivity during titration. The precision and the accuracy of the developed method was established by the analysis of quality control samples; %RSD of corrected conductance values &lt;2% and recovery results within 100 ± 2% were achieved. The calibration graphs obtained were linear over the concentrations 1.0–1.4 mM for all the drugs (R2&gt; 0.99). The drugs were successfully analyzed in their respective dosage forms prepared in-house. The method has offered easier, faster and cost-effective analysis of the selected drugs and can be used for routine determinations in the quality control laboratories. More importantly, it is an environmental friendly procedure, as no organic solvent was used throughout the analysis.

Concepts and Software Package for Efficient Quality Control in Targeted Metabolomics Studies: MeTaQuaC.

Targeted quantitative mass spectrometry metabolite profiling is the workhorse of metabolomics research. Robust and reproducible data are essential for confidence in analytical results and are particularly important with large-scale studies. Commercial kits are now available which use carefully calibrated and validated internal and external standards to provide such reliability. However, they are still subject to processing and technical errors in their use and should be subject to a laboratory's routine quality assurance and quality control measures to maintain confidence in the results. We discuss important systematic and random measurement errors when using these kits and suggest measures to detect and quantify them. We demonstrate how wider analysis of the entire data set alongside standard analyses of quality control samples can be used to identify outliers and quantify systematic trends to improve downstream analysis. Finally, we present the MeTaQuaC software which implements the above concepts and methods for Biocrates kits and other target data sets and creates a comprehensive quality control report containing rich visualization and informative scores and summary statistics. Preliminary unsupervised multivariate analysis methods are also included to provide rapid insight into study variables and groups. MeTaQuaC is provided as an open source R package under a permissive MIT license and includes detailed user documentation.

LC-MS Quantification of Malondialdehyde-Dansylhydrazine Derivatives in Urine and Serum Samples.

We developed a robust analytical method for quantification of malondialdehyde (MDA) in urine and serum samples using dansylhydrazine (DH) as a derivatizing reagent. The derivatization procedure was partially carried out using an autosampler injection program to minimize errors associated with the low-volume addition of reagents and was optimized to yield a stable hydrazone derivative of MDA and its labeled d2-MDA analogue. The target MDA-DH derivatives were separated on an Agilent Zorbax Eclipse Plus Phenyl-Hexyl (3.0×100mm, 3.5μm) column. The mass-to-charge ratios of the target derivatives [(M+H)+ of 302 and 304 for MDA-DH and d2-MDA-DH, respectively] were analyzed in single ion monitoring mode using a single quadrupole mass spectrometer operated under positive electrospray ionization. The method limits of quantification were 5.63nM (or 0.405ng/mL) for urine analysis and 5.68nM (or 0.409ng/mL) for serum analysis. The quantification range for urine analysis was 5.63-500nM (0.405-36.0ng/mL) while the quantification range for serum analysis was 5.68-341nM (0.409-24.6ng/mL). The method showed good relative recoveries (98-103%), good accuracies (92-98%), and acceptable precisions (relative standard deviations 1.8-7.3% for inter-day precision; 1.8-6.1% for intra-day precision) as observed from the repeat analysis of quality control samples prepared at different concentrations. The method was used to measure MDA in individual urine samples (n=287) and de-identified archived serum samples (n=22) to assess the overall performance of the method. The results demonstrated that our method is capable of measuring urinary and serum levels of MDA, allowing its future application in epidemiologic investigations.

Rapid and Sensitive Quantification of Ursolic Acid and Oleanolic Acid in Human Plasma Using Ultra-performance Liquid Chromatography-Mass Spectrometry.

Ultra-performance liquid chromatography (UPLC) interfaced with atmospheric pressure chemical ionization mass-spectrometry was used to separate and quantify ursolic acid (UA) and oleanolic acid (OA) in human plasma. UA and OA were extracted from 0.5 mL human plasma using supported liquid extraction and separated utilizing an Acquity UPLC HSS column. The method has been validated for both UA and OA quantitation with a limit of detection of 0.5 ng/mL. The UPLC separations are carried out with isocratic elution with methanol and 5 mM ammonium acetate in water (85:15) as a mobile phase at a flow rate of 0.4 mL/min. The assay was linear from 1 ng/mL to 100 ng/mL for both analytes. The total analysis time was 7 min with the retention times of 3.25 (internal standard), 3.65 (UA) and 3.85 min (OA). Recovery of drug from plasma ranged from 70% to 115%. Analysis of quality control samples at 3, 30 and 80 ng/mL (n = 14) had an intra-day coefficient of variation of 9.9%, 4.3% and 5.5%, respectively. A proof-of-concept study in human patients who consumed apple peels indicates that this analytical method could be applied to clinical studies of UA and/or OA in human subjects.

Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry.

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150 × 4.6 mm, 5 μm) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8 mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2 mL/min. The total chromatographic run time was 10 min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.08 → 72.09 for metoprolol and m/z 303.3 → 154.3 for IS. The linearity range was 2.5-500 ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5 ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories. Copyright © 2016 John Wiley & Sons, Ltd.

Study of circulating IgG antibodies to peptide antigens derived from BIRC5 and MYC in cervical cancer

The present study was undertaken to detect circulating IgG antibodies to peptide antigens derived from baculoviral IAP repeat-containing protein 5 isoform 2 (BIRC5) and myc proto-oncogene protein (MYC) in cervical cancer. A total of 107 female patients with cervical cancer of stages I and II, and 130 healthy female subjects were recruited for analysis of circulating IgG antibodies to BIRC5 and MYC. Student’s t-test showed significant differences in circulating levels of anti-BIRC5 IgG (t=−4.27, df=235, P<0.0001) and anti-MYC IgG (t=3.51, df=232, P=0.0005) between the patient group and the control group. Receiver operating characteristic (ROC) analysis showed an area under the ROC curve (AUC) of 0.67 with sensitivity of 23.4% against specificity of 90% for the anti-BIRC5 IgG assay and an AUC of 0.66 with sensitivity of 9.4% against specificity of 90.6% for the anti-MYC IgG assay. Analysis of quality control samples gave an inter-assay deviation of 8.9% in the anti-BIRC5 IgG assay and 9.0% in the anti-MYC IgG assay. This work suggests that anti-BIRC5 IgG could serve as a biomarker for early diagnosis of cervical cancer although a panel of such tumor-associated antigens is needed to develop a highly sensitive test.

Liquid chromatography–mass spectrometry analysis of diethylcarbamazine in human plasma for clinical pharmacokinetic studies

A sensitive and selective liquid chromatographic method using mass spectrometric detection was developed for the determination of diethylcarbamazine (DEC) in human plasma. DEC and its stable isotope internal standard d3-DEC were extracted from 0.25mL of human plasma using solid phase extraction. Chromatography was performed using a Phenomenex Synergi 4μ Fusion-RP column (2mm×250mm) with gradient elution. The retention time was approximately 4.8min. The assay was linear from 4 to 2200ng/mL. Analysis of quality control samples at 12, 300, and 1700ng/mL (N=15) had interday coefficients of variation of 8.4%, 5.4%, and 6.2%, respectively (N=15). Interday bias results were −2.2%, 6.0%, and 0.8%, respectively. Recovery of DEC from plasma ranged from 84.2% to 90.1%. The method was successfully applied to clinical samples from patients with lymphatic filariasis from a drug–drug interaction study between DEC and albendazole and/or ivermectin.

Circulating IgG antibody against FOXP3 may be a potential biomarker for lung cancer

Background: Forkhead box P3 (FOXP3) has been found to be overexpressed by a range of cancer cells and correlated with prognosis of tumors. This finding raises the possibility that the development of anti-FOXP3 antibody test may be useful for clinical application. Objective: The present work was designed to test whether circulating autoantibody to FOXP3 was altered in lung cancer. Methods: 271 patients with non-small cell lung cancer (NSCLC) and 227 in control subjects matched in age, gender and smoking history were recruited. Circulating anti-FOXP3 IgG antibody was tested using an in-house enzymelinked immunosorbent assay. Results: Student’s t-test showed that the levels of IgG autoantibody to FOXP3 were significantly higher in patients with NSCLC than control subjects (t = 7.67, P 0.65 - 0.75, SE ± 0.024), in which patients at stage 2 had the highest AUC of 0.75 (95%CI 0.67 - 0.81, SE ± 0.037), with a sensitivity of 31.4% against a specificity of 90.3%. Analysis of quality control samples gave an inter-assay deviation of 13.3% among 45 plates tested. Conclusions: Circulating IgG autoantibody to FOXP3 may be a potential biomarker for lung cancer.

A rapid and sensitive gas chromatography-mass spectrometry method for the quality control of perfumes: simultaneous determination of phthalates

A rapid and sensitive analytical gas chromatography-mass spectrometry (GC-MS) method for perfume analysis to determine the phthalates banned by the European Union Regulation on cosmetic samples is presented. This method has been tested in commercial alcoholic perfume samples for the determination of the following seven phthalates: dibutyl phthalate, bis(2-ethylhexyl) phthalate, bis(2-methoxyethyl) phthalate, n-pentyl-isopentylphthalate, di-n-pentyl phthalate, diisopentylphthalate and benzyl butyl phthalate. Sample evaporation and redissolution in ethanol is carried out before GC-MS analysis, with no dilution of the sample. External calibration and standard addition calibration are compared to detect possible matrix effects. External calibration could provide good results in some cases but standard addition calibration is recommended as matrix effects are observed in many cases in the determination of the target phthalates. The accuracy of the method has been proven by the analysis of quality control samples prepared from commercial free phthalate perfumes after spiking with known concentrations of the analytes. Accurate results were obtained with limits of detection in the ng mL−1 range and good repeatability (relative standard deviations lower than 6%). The method was satisfactorily applied to the analysis of commercial perfume samples, and the results revealed considerable amounts of dibutyl phthalate and bis(2-ethylhexyl) phthalate in some of the analyzed samples.

Improvements in the analytical methodology for the residue determination of the herbicide glyphosate in soils by liquid chromatography coupled to mass spectrometry

The determination of glyphosate (GLY) in soils is of great interest due to the widespread use of this herbicide and the need of assessing its impact on the soil/water environment. However, its residue determination is very problematic especially in soils with high organic matter content, where strong interferences are normally observed, and because of the particular physico-chemical characteristics of this polar/ionic herbicide. In the present work, we have improved previous LC–MS/MS analytical methodology reported for GLY and its main metabolite AMPA in order to be applied to “difficult” soils, like those commonly found in South-America, where this herbicide is extensively used in large areas devoted to soya or maize, among other crops. The method is based on derivatization with FMOC followed by LC–MS/MS analysis, using triple quadrupole. After extraction with potassium hydroxide, a combination of extract dilution, adjustment to appropriate pH, and solid phase extraction (SPE) clean-up was applied to minimize the strong interferences observed. Despite the clean-up performed, the use of isotope labelled glyphosate as internal standard (ILIS) was necessary for the correction of matrix effects and to compensate for any error occurring during sample processing. The analytical methodology was satisfactorily validated in four soils from Colombia and Argentina fortified at 0.5 and 5mg/kg. In contrast to most LC–MS/MS methods, where the acquisition of two transitions is recommended, monitoring all available transitions was required for confirmation of positive samples, as some of them were interfered by unknown soil components. This was observed not only for GLY and AMPA but also for the ILIS. Analysis by QTOF MS was useful to confirm the presence of interferent compounds that shared the same nominal mass of analytes as well as some of their main product ions. Therefore, the selection of specific transitions was crucial to avoid interferences. The methodology developed was applied to the analysis of 26 soils from different areas of Colombia and Argentina, and the method robustness was demonstrated by analysis of quality control samples along 4 months.

Development and validation of a LC‐MS/MS assay to quantify intracellular tenofovir‐diphosphate (TFV‐DP) and emtricitabine‐triphosphate (FTC‐TP)

IntroductionCombination antiretroviral therapy has been associated with dramatic reductions in morbidity and mortality in HIV‐infected patients. The key to successful HIV treatment is strict adherence to the prescribed regimen. The combination of tenofovir, emtricitabine and efavirenz co‐formulated in a single tablet (Atripla) provides a ‘one tablet once a day’ regimen, making adherence easier for patients. Tenofovir and emtricitabine are converted intracellularly to active phosphate anabolites. Therefore, knowledge of intracellular pharmacokinetics should aid understanding of the virological response and toxicity of these agents. This intracellular methodology has been developed and applied to clinical trial samples.MethodPeripheral blood mononuclear cells (PBMC) were isolated from whole blood by gradient centrifugation using ficoll. PBMC were counted and lysed (70% MeOH) giving a final cell density of 2×106 cells/ml. Lysate was centrifuged, supernatant (intracellular matrix), was spiked with drug anabolites at various concentrations to produce standard curves and quality controls (QC). Samples were extracted by protein precipitation (acetonitrile), evaporated to dryness, internal standard (IS) added, and reconstituted in 5 mM ammonuim formate. Weak anion exchange chromatography with an optimised step‐wise gradient, interfaced with a triple quadrupole mass spectrometer operated in SRM with positive ionisation mode was used for quantification.ResultsAnalytes eluted within 12 minutes run‐time with adequate separation. Calibration curves were validated over the following range TFV‐DP = 0.35–10.91 ng/mL, FTC‐TP = 0.38–103.17 ng/mL (r2 values &gt; 0.99; linear 1/x). The lower limit of quantification was &lt; 20%, signal to noise was &gt; 5% and carryover &lt; 0.1%. The precision (relative standard deviation,% RSD) of the assays as determined from analysis of quality control samples was; TFV‐DP = 6.3–11% and FTC‐TP = 6–18.6%, and accuracy was TFV‐DP = 97.5–100.8%, FTC‐TP = 98–100.3%.ConclusionsA direct, highly sensitive intracellular anabolite assay has been developed and validated which elutes analytes and IS rapidly and has been applied to patient samples from a pivotal trial the results of which are crucial in our understanding of antiretroviral drug ‘forgiveness’.

Everolimus quantification in peripheral blood mononuclear cells using ultra high performance liquid chromatography tandem mass spectrometry

A reliable ultra high performance liquid chromatography tandem mass spectrometry method has been developed for the determination of everolimus in human peripheral blood mononuclear cells (PBMCs). Protein precipitation was used for sample preparation. Analysis was performed on an UPLC Waters Acquity system. Chromatography was carried out using a cartridge column MassTrak TDM C18 (2.1×10 mm) with 0.1% formic acid and 2 mM of ammonium acetate in water and 0.1% formic acid and 2mM of ammonium acetate in methanol mixture as a mobile phase delivered at a flow rate of 0.4 mL/min in gradient mode. The assay was linear over a range of 0-12.5 ng/mL. The analysis of quality control samples at 2.5, 5.0 and 10.0 ng/mL demonstrated good precision with relative standard deviation of less than 15%. Recoveries at concentrations of 2.5, 5.0 and 10.0 ng/mL were all greater than 83%. The method was successfully applied to the analysis of everolimus in PBMCs from blood samples of transplant recipients.

Development and validation of a bioanalytical LC–MS method for the quantification of GHRP-6 in human plasma

Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH 2, MW = 872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with 13C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC–MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R 2 higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial.

Direct aqueous injection with backflush thermal desorption for wastewater monitoring by online GC-MS

A gas chromatography-mass spectrometry system with a novel injector type, which is designed for direct aqueous injection of wastewater, is presented. The system is used for online monitoring of the influent of the wastewater treatment plant at BASF's main chemical production site in Ludwigshafen, Germany. The purpose of monitoring is to protect the biological treatment process and the receiving water body, the Rhine. The modular system is primarily based on commercial equipment, but utilizes a special injection system, which is connected to a Deans switch. The two-stage injector consists of a programmable temperature vaporizer (PTV) injector with a small volume insert for vaporization and a dual sorbent packed second PTV for analyte adsorption/desorption. The Deans switch allows a backflush/thermal desorption operation which enables the direct injection of filtered, crude wastewater. About 170 volatile and semivolatile compounds are calibrated with approximate detection limits of 1 mg/L, which are sufficient for the analysis of untreated wastewater. The quantitative results are transferred to a database which is connected to a process control system. If the wastewater does not meet the required specification, an alarm is generated and the wastewater is diverted into a storage basin. Special software programs and routines allow for reliable, unattended operation and remote instrument control. Data quality is automatically controlled in each run and through the daily analysis of quality control samples. The current design allows for analysis of volatile compounds, such as methanol, whereas an earlier injector setup restricted the range of analytes to less volatile compounds (of size C(4) or greater).

Simultaneous Measurement of 17β-Estradiol, 17α-Estradiol and Estrone by GC-Isotope Dilution MS/MS.

A convenient GC-MS/MS-based method was developed for the simultaneous measurement of 17β-estradiol, 17α-estradiol and estrone using liquid-liquid extraction, a single-step derivatization with N-(trimethylsilyl)imidazole and the corresponding deuterated estrogens as internal standards. Separation of these estrogens was achieved on a 50% phenyl polysilphenylene-siloxane bonded phase column. MS/MS response factors for the derivatized analytes and their corresponding internal standards were found to be practically identical. Therefore, analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with known molar concentration of the corresponding deuterated internal standards. Assay accuracies, determined from the analyses of quality control samples obtained by spiking known concentrations of analytes into charcoal-stripped human serum, were in the -11% to +14% range. Limits of quantitations were between 13 pg/mL and 21 pg/mL from this biological medium.