Simultaneous quantification of total eicosapentaenoic acid, docosahexaenoic acid and arachidonic acid in plasma by high-performance liquid chromatography-tandem mass spectrometry.

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid-liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r(2) > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30-90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega-3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre-treatment concentrations varied between subjects for EPA (17-68 mg/L), DHA (36-63 mg/L) and AA (121-248 mg/L). During the dosing period EPA increased 460-480% from the baseline concentration, while DHA increased 150-160%. The EPA-AA ratio increased from 0.07-0.56 to 0.3-3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death.

Liquid chromatography-tandem mass spectrometry method for the simultaneous quantitative determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood.

Simultaneous determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood by HPLC-tandem mass spectrometry was developed and validated. The pesticides were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Luna C(18) (30mmx2.0mm, 3microm) column, using a step-gradient at a flow rate of 0.4ml/min. The assay was linear from 0.5 to 100ng/ml (r(2)>0.992, n=24) for all pesticides. The inter- and intra-day accuracy and precision for the method was 96.6-106.1% and <10%, respectively. The lower limit of quantification was 0.5ng/ml. In conclusion, the method described displays analytical performance characteristics that are suitable for the quantification of these pesticides in cases of acute poisoning.

A high-performance liquid chromatography-mass spectrometry method using a novel atmospheric pressure chemical ionization approach for the rapid simultaneous measurement of tacrolimus and cyclosporin in whole blood.

Concentration monitoring and dose individualization is required to optimize either tacrolimus or cyclosporin therapy. In this study, the validation of a simple, rapid high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous measurement of tacrolimus and cyclosporin in whole blood is reported. Blood samples (100 microL) were prepared by protein precipitation with zinc sulphate followed by acetonitrile (containing the internal standards ascomycin and cyclosporin D). The chromatographic run time was 1.5 minutes per sample. Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in negative ionization mode (tacrolimus: m/z 802.5 --> 560.6, cyclosporin: m/z 1200.8 --> 1088.4). The assay had an analytical range of 1.0 to 30 mug/L (r > 0.998, n = 6) for tacrolimus and 25 to 2000 microg/L (r > 0.999, n = 6) for cyclosporin. Tacrolimus inter- and intraday inaccuracy and precision [coefficient of variation (CV)] using quality control samples (2.5, 12.5, 25 microg/L) was less than +/-10.0% and CV less than 5.0%, respectively (n = 5). Similarly, cyclosporin inter- and intraday inaccuracy and precision using quality control samples (70, 400, 1500 microg/L) was less than +/-2.0% and CV less than 5.0%, respectively (n = 5). The lower limit of quantification for tacrolimus was 1.0 mug/L and cyclosporin 25 microg/L. The assay had an absolute mean recovery of 86.7% for tacrolimus and 89.0% for cyclosporin (n = 15). Intersubject variability, as a measure of potential matrix effects on results, was less than 6.0% CV for both analytes (n = 15). Extracted samples were stable for at least 20 hours. Results obtained from external proficiency testing samples measured by this method compared with the mean of all liquid chromatography-tandem mass spectrometry methods used by scheme participants revealed a strong correlation and good agreement for tacrolimus (r = 0.993, mean bias = -10.3%, n = 19) and cyclosporin (r = 0.996, mean bias = 3.0%, n = 20). In conclusion, this is the first reported high-throughput method that uses negative atmospheric pressure chemical ionization for the simultaneous measurement of tacrolimus and cyclosporin in whole blood.

Cloned enzyme donor immunoassay tacrolimus assay compared with high-performance liquid chromatography-tandem mass spectrometry and microparticle enzyme immunoassay in liver and renal transplant recipients.

The immunosuppressant drug tacrolimus has a narrow therapeutic index and is subject to a large variation in individual bioavailability and clearance. With its narrow therapeutic index, therapeutic drug monitoring is standard clinical practice in the management of transplant recipients. In this study, we report the evaluation of the cloned enzyme donor immunoassay (CEDIA) for the determination of whole-blood tacrolimus concentrations compared with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and microparticle enzyme immunoassay (MEIA) using samples obtained from liver (n = 100) and renal (n = 88) transplant recipients. Linear regression analysis showed a relationship of CEDIA = 1.24 HPLC-MS/MS -0.18 (r = 0.81). The mean bias (+/-SEM) for all patients when compared with HPLC-MS/MS was 22.2% (+/-2.1%). The precision of the CEDIA method for all samples showed a root mean square error of 3.1 microg/L. Liver transplant recipient samples showed a mean (+/-SEM) bias compared with HPLC-MS/MS of 12.5% (+/-1.6%). The precision of the CEDIA method for these samples showed a root mean square error of 1.5 microg/L. The data suggest that in the renal transplant group, the CEDIA and MEIA methods have a bias of 33.3% and 20.1%, respectively, compared with HPLC-MS/MS. The CEDIA tacrolimus immunoassay has been shown to be a rapid method for the determination of whole-blood tacrolimus concentrations and may be considered when HPLC-MS/MS is not available. When used in the clinical setting with other parameters, it would be a useful adjunct in the management of liver transplant recipients, but a significant bias in renal transplant patients needs to be further investigated.

FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development of atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: Numerous in vitro studies suggest that sphingosine 1-phosphate (S1P), a bioactive lysosphingolipid associated with high-density lipoproteins, accounts at least partly for the potent antiinflammatory properties of high-density lipoprotein and, thereby, contributes to the antiatherogenic potential attributed to high-density lipoproteins. The present study was undertaken to investigate whether modulation of S1P signaling would affect atherosclerosis in a murine model of disease. METHODS AND RESULTS: Low-density lipoprotein receptor-deficient mice on a cholesterol-rich diet were given FTY720, a synthetic S1P analogue, at low (0.04 mg/kg per day) or high (0.4 mg/kg per day) doses for 16 weeks. FTY720 dose-dependently reduced atherosclerotic lesion formation, both in the aortic root and brachiocephalic artery, and almost completely blunted necrotic core formation. Plasma lipids remained unchanged during the course of FTY720 treatment. However, FTY720 lowered blood lymphocyte count (at a high dose) and significantly interfered with lymphocyte function, as evidenced by reduced splenocyte proliferation and interferon-gamma levels in plasma. Plasma concentrations of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and regulated on activation normal T cell expressed and secreted were reduced by FTY720 administration. Moreover, lipopolysaccharide-elicited generation of nitrite/nitrate and IL-6--two markers of classical (M1) macrophage activation--was inhibited, whereas IL-4-induced production of IL-1-receptor antagonist, a marker of alternative (M2) macrophage activation, was augmented in peritoneal macrophages from FTY720-treated low-density lipoprotein receptor-deficient mice. CONCLUSIONS: The present results demonstrate that an S1P analogue inhibits atherosclerosis by modulating lymphocyte and macrophage function, and these results are consistent with the notion that S1P contributes to the antiatherogenic potential of high-density lipoprotein.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3-->255.3). The assay was linear from 0.2 to 25 microg/l (r(2)>0.997, n=5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 microg/l) were 95.8-103.2 and <5.5%, respectively. At the lower limit of quantification (0.2 microg/l) the inter- and intra-day analytical recovery was 99.0-102.8% with imprecision of <7.6% (n=5). The assay had a mean relative recovery of 100.5+/-5.8% (n=15). Extracted samples were stable for 16 h. FTY720 quality control samples were stable at room temperature for 16 h, at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans.

Evaluation of a fluorescent polarization immunoassay for whole blood everolimus determination using samples from renal transplant recipients.

OBJECTIVES: This study compared the performance of a fluorescent polarization immunoassay (FPIA) against HPLC-tandem mass spectrometry (HPLC-MS) for the measurement of everolimus in renal transplant recipients. DESIGN AND METHODS: A total of 333 pre-dose samples from 45 renal transplant patients were analyzed by FPIA and HPLC-MS. RESULTS: The inter-batch inaccuracy and precision of the FPIA for control samples were

Comparison of the reintroduced MEIA assay with HPLC-MS/MS for the determination of whole-blood sirolimus from transplant recipients.

Therapeutic monitoring with dosage individualization of sirolimus drug therapy is standard clinical practice for organ transplant recipients. For several years sirolimus monitoring has been restricted as a result of lack of an immunoassay. The recent reintroduction of the microparticle enzyme immunoassay (MEIA) for sirolimus on the IMx analyser has the potential to address this situation. This study, using patient samples, has compared the MEIA sirolimus method with an established HPLC-tandem mass spectrometry method (HPLC-MS/MS). An established HPLC-UV assay was used for independent cross-validation. For quality control materials (5, 11, 22 microg/L), the MEIA showed acceptable validation criteria based on intra- and inter-run precision (CV) and accuracy (bias) of <8% and <13%, respectively. The lower limit of quantitation was found to be approximately 3 microg/L. The performance of the immunoassay was compared with HPLC-MS/MS using EDTA whole-blood samples obtained from various types of organ transplant recipients (n = 116). The resultant Deming regression line was: MEIA =1.3 x HPLC-MS/MS + 1.3 (r = 0.967, S(y/x) = 1) with a mean bias of 49.2% +/- 23.1% (range, -2.4% to 128%; P<0.001). The reason for the large and variable bias was not explored in this study, but the sirolimus-metabolite cross-reactivity with the MEIA antibody could be a substantive contributing factor. Whereas the MEIA sirolimus method may be an adjunct to sirolimus dosage individualization in transplant recipients, users must consider the implications of the substantial and variable bias when interpreting results. In selected patients where difficult clinical issues arise, reference to a specific chromatographic method may be required.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices.

OBJECTIVES: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. METHODS: Blood samples (50 muL) were prepared by protein precipitation followed by C(18) solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. RESULTS: The assay was linear from 10 to 2000 microg/L (r(2)>0.996, n=9). Inter-day analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 microg/L were 94.9--103.5% and <7.7%, respectively (n=5). The assay had a mean relative recovery of 101.6%. Ion suppression was<8.0% of the total signal (n=15). Extracted samples were stable for 6 h. Patient samples, measured by this method and compared with a validated HPLC-UV assay, revealed a strong correlation (r=0.998) and excellent agreement with a mean percentage bias of 2.1% (n=60). CONCLUSION: This high-throughput method provides accurate, precise, and specific measurement of cyclosporin in blood over a wide analytical range, thus making it suitable for current clinical monitoring strategies.

CEDIA sirolimus assay compared with HPLC-MS/MS and HPLC-UV in transplant recipient specimens.

The role of the therapeutic drug monitoring laboratory in support of immunosuppressant drug therapy is well established, and the introduction of sirolimus (SRL) is a new direction in this field. The lack of an immunoassay for several years has restricted the availability of SRL assay services. The recent availability of a CEDIA SRL assay has the potential to improve this situation. The present communication has compared the CEDIA SRL method with 2 established chromatographic methods, HPLC-UV and HPLC-MS/MS. The CEDIA method, run on a Hitachi 917 analyzer, showed acceptable validation criteria with within-assay precision of 9.1% and 3.3%, and bias of 17.1% and 5.8%, at SRL concentrations of 5.0 microg/L and 20 microg/L, respectively. The corresponding between-run precision values were 11.5% and 3.3% and bias of 7.1% and 2.9% at 5.0 microg/L and 20 microg/L, respectively. The lower limit of quantification was found to be 3.0 microg/L. A series of 96 EDTA whole-blood samples predominantly from renal transplant recipients were assayed by the 3 methods for comparison. It was found that the CEDIA method showed a Deming regression line of CEDIA=1.20xHPLC-MS/MS-0.07 (r=0.934, SEE=.47), with a mean bias of 20.4%. Serial blood samples from 8 patients included in this evaluation showed that the CEDIA method reflected the clinical fluctuations in the chromatographic methods, albeit with the variable bias noted. The CEDIA method on the H917 analyzer is therefore a useful adjunct to SRL dosage individualization in renal transplant recipients.

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry.

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were prepared by protein precipitation, followed by C(18) solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 microg/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 microg/l, was 100.3 - 105.4% and < or = 7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degrees C for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase II and III clinical trials.