Internal Standard Solution Research Articles

Automated saliva processing for LC-MS/MS: Improving laboratory efficiency in cortisol and cortisone testing

ObjectivesThe aim of this study was to implement in our routine practice an automated saliva preparation protocol for quantification of cortisol (F) and cortisone (E) by LC-MS/MS using a liquid handling platform, maintaining the previously defined reference intervals with the manual preparation. Design and methodsAddition of internal standard solution to saliva samples and calibrators and SPE on μ-elution 96-well plate were performed by liquid handling platform. After extraction, the eluates were submitted to LC-MS/MS analysis. The manual steps within the entire process were to transfer saliva samples in suitable tubes, to put the cap mat and transfer of the collection plate to the LC auto sampler. Transference of the reference intervals from the manual to the automated procedure was established by Passing Bablok regression on 120 saliva samples analyzed simultaneously with the two procedures. ResultsCalibration curves were linear throughout the selected ranges. The imprecision ranged from 2 to 10%, with recoveries from 95 to 116%. Passing Bablok regression demonstrated no significant bias. ConclusionsThe liquid handling platform translates the manual steps into automated operations allowing for saving hands-on time, while maintaining assay reproducibility and ensuring reliability of results, making it implementable in our routine with the previous established reference intervals.

Rapid determination of residues of pesticides in honey by µGC-ECD and GC-MS/MS: Method validation and estimation of measurement uncertainty according to document No. SANCO/12571/2013

ABSTRACTA simple and straightforward method for simultaneous determination of residues of 13 pesticides in honey samples (acrinathrin, bifenthrin, bromopropylate, cyhalothrin-lambda, cypermethrin, chlorfenvinphos, chlorpyrifos, coumaphos, deltamethrin, fluvalinate-tau, malathion, permethrin and tetradifon) from different pesticide classes has been developed and validated. The analytical method provides dissolution of honey in water and an extraction of pesticide residues by n-Hexane followed by clean-up on a Florisil SPE column. The extract was evaporated and taken up by a solution of an injection internal standard (I-IS), ethion, and finally analyzed by capillary gas chromatography with electron capture detection (GC-µECD). Identification for qualitative purpose was conducted by gas chromatography with triple quadrupole mass spectrometer (GC-MS/MS). A matrix-matched calibration curve was performed for quantitative purposes by plotting the area ratio (analyte/I-IS) against concentration using a GC-µECD instrument. According to document No. SANCO/12571/2013, the method was validated by testing the following parameters: linearity, matrix effect, specificity, precision, trueness (bias) and measurement uncertainty. The analytical process was validated analyzing blank honey samples spiked at levels equal to and greater than 0.010 mg/kg (limit of quantification). All parameters were satisfactorily compared with the values established by document No. SANCO/12571/2013. The analytical performance was verified by participating in eight multi-residue proficiency tests organized by BIPEA, obtaining satisfactory z-scores in all 70 determinations. Measurement uncertainty was estimated according to the top-down approaches described in Appendix C of the SANCO document using the within-laboratory reproducibility relative standard deviation combined with laboratory bias using the proficiency test data.

Pre-concentration of chloride in dilute water-samples for precise δ37Cl determination using a strong ion-exchange resin: Application to rainwaters

Low chloride concentration in water samples is a limitation for chlorine stable isotope measurements using a gas source isotope ratio mass spectrometer (IRMS). Here we describe an optimized method of chloride (Cl−) pre-concentration for dilute waters using a strong anion-exchange resin (Amberlite AG 1-X4). In order to validate our method and its reliability for δ37Cl measurements we made high precision δ37Cl measurements by dual inlet (DI) IRMS, also applicable to δ37Cl measurements by continuous flow IRMS.A 3ml solid phase extraction (SPE) cartridge is filled with 1ml of Amberlite resin in hydroxide form (100–200mesh) and is rinsed with 50ml of a 1M NaOH solution. The water sample containing aqueous chlorides is then loaded through the column at a flow rate of about 1.5ml/min. Because of the high partition coefficient of Cl− between resin and water (Kd>5300) the exchange of Cl− onto the resin is very efficient. We found that a volume as low as 5ml of a 1M KNO3 solution is enough for quantitative Cl− recovery from 1ml of resin. Using an internal laboratory standard solution as well as natural samples (seawaters and hydrothermal waters) with more complex chemistries and known δ37Cl values, the efficiency of this method was validated in terms of Cl− recovery yields, as well as accuracy and reproducibility of δ37Cl measurements.For dilute standard solutions with chloride concentrations ranging from 0.02 to 0.4mmol/l (Cl amounts ranging from 20 to 80μmol), the whole procedure recovery yields (ie. including the steps of Cl− pre-concentration and conversion into CH3Cl as well as consecutive CH3Cl purification) are 101±7% (1σ, n=21) and the measured δ37Cl agrees with the expected δ37Cl value to within ±0.04‰ (1σ), our current level of external precision reached by DI-IRMS measurements. Seawaters and hydrothermal waters with known δ37Cl values have also been diluted and treated using this method. All samples show quantitative Cl− recovery (yields~100%) and accurate δ37Cl values, with one maximum shift from the expected value of +0.09‰. We also found that for water samples containing relatively high concentrations of anions with a higher affinity for the resin than chlorides, such as sulfates or nitrates, the resin capacity may be exceeded, preventing a quantitative recovery of Cl− and yielding lower δ37Cl than expected. For such samples, we recommend increasing the volume of resin adequately. This method allows the investigation of as yet poorly documented δ37Cl in dilute natural waters (e.g., soil solutions, rainwaters and river waters) with highly precise DI-IRMS measurements. In a preliminary investigation, we applied our method to 6 rainwater samples collected at the summit and lowland of the Soufrière volcano. For Cl− concentrations between 0.06 and 0.1mmol/l we found small but significant δ37Cl variations, with values ranging from −0.16 to +0.38‰ (±0.04‰, 1σ). These results demonstrate that in active volcanic contexts, where rainwater Cl− may be partly sourced from volcanic degassing, δ37Cl carries information, which deserves further investigation given its potential implication for volcanic monitoring.

Rapid, easy analysis of urinary vanillylmandelic acid for diagnostic testing of pheochromocytoma by liquid chromatography tandem mass spectrometry.

Vanillylmandelic acid (VMA), as one of the most important catecholamine metabolites, is commonly used to aid in diagnosis of pheochromocytoma. This study develops a rapid and simple high-throughput LC-MS/MS method for the measurement of urinary VMA. Without sample pretreatment, the urine specimens were mixed with internal standard (IS) solution for direct analysis by LC-MS/MS in two minutes. VMA and VMA-d3 were detected in the multiple-reaction monitoring mode using the specific transitions m/z 197.0→137.0 and 200.0→140.0, respectively. This method was validated for consistent linearity from 1.0 to 250.0μM with CVs≤3.12%, excellent recovery, good stability and low carryover. The lowest limit of quantification (LLOQ) was 0.125μmol/L for VMA with CV of 14.1%, and the lower limit of detection (LOD) was 0.025μmol/L. Intra-assay and inter-assay imprecision values (CVs) for VMA were all below 2.11%. Dilution linearity was investigated with a satisfied mean accordance of 105%. Method comparison of LC-MS/MS and microcolumn chromatography in our lab was performed and the reference interval was established in agreement with that of the Mayo Clinic. All these results demonstrate that this validated LC-MS/MS approach shows improved accuracy and reproducibility compared with microcolumn chromatography. The low sample volume, simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.

Enantiomer Profiling of Methamphetamine in White Crystal and Tablet Forms (Ma Old) Using LC-MS-MS.

At the global level, seizures of crystalline methamphetamine (MA) and MA tablets have risen to a new high, indicating that the substance is an imminent threat. MA enantiomer profiling was a useful tool to investigate the prevalence of MA abuse, the intrinsic characteristics of the seized samples and the trends of precursors. In this work, the distribution of enantiomers in clandestine MA crystals and tablets seized mainly in the Yangtze River Delta region, China, from 2008 to 2014 were identified. The MA samples were diluted with internal standard methanol solution, and analyzed by LC-MS-MS. The detection limits of the enantiomers were 0.04 μg/L. The limit of quantification was 0.1 μg/L. As little as 0.2% of the R-enantiomer ratio could be determined. Standard calibration curves of S- and R-MA showed good linearity in the range of 0.1-80 μg/L (r(2) > 0.995). All of the seizures were optically pure S-enantiomer in the years 2008 and 2009. Seized samples containing a slight amount of R-MA began to appear in 2010 and increased in the year 2014. No racemic mixture or R-isomer of MA was seized. From this study, we could find out that smuggling routes and/or precursors might be silently changing in the Yangtze River Delta region, China.

Evaluation of a commercial LC-MS/MS assay for the quantification of ethyl glucuronide in urine for alcohol intake monitoring

Abstract Background: A commercially available in vitro diagnostics (IVD)-approved mass spectrometric assay for the quantification of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine (RECIPE Chemicals+Instruments GmbH, Munich, Germany) was verified for monitoring of recent alcohol intake after transplantation. Methods: For sample preparation, 50 μL of urine sample was mixed with an isotope-labeled internal standard solution. After centrifugation, 5 μL of the supernatant was analyzed by LC-MS/MS in a total run time of 3 min. An API 6500 tandem mass spectrometer (AB SCIEX, Toronto, Canada) combined with a Shimadzu UFLC system (Duisburg, Germany) was applied. Results: The limits of quantification for the commercial assay were 0.07 mg/L for EtG and 0.03 mg/L for EtS in urine. The coefficient of variation for both analytes was lower than 7% (within-day) and 15% (between-days). Accuracy ranged between 101 and 144% for samples from an external quality assurance program. The comparison of the commercial test kit and an established LC-MS/MS method showed a very good agreement for EtG (r=0.96) and EtS (r=0.97) over a broad urine concentration range. Conclusions: The commercial IVD-certified LC-MS/MS assay is suitable for the analysis of EtG and EtS in human urine[0] to assess recent alcohol intake in transplant monitoring.

Green approach for ultratrace determination of divalent metal ions and arsenic species using total-reflection X-ray fluorescence spectrometry and mercapto-modified graphene oxide nanosheets as a novel adsorbent.

A new method based on dispersive microsolid phase extraction (DMSPE) and total-reflection X-ray fluorescence spectrometry (TXRF) is proposed for multielemental ultratrace determination of heavy metal ions and arsenic species. In the developed methodology, the crucial issue is a novel adsorbent synthesized by grafting 3-mercaptopropyl trimethoxysilane on a graphene oxide (GO) surface. Mercapto-modified graphene oxide (GO-SH) can be applied in quantitative adsorption of cobalt, nickel, copper, cadmium, and lead ions. Moreover, GO-SH demonstrates selectivity toward arsenite in the presence of arsenate. Due to such features of GO-SH nanosheets as wrinkled structure and excellent dispersibility in water, GO-SH seems to be ideal for fast and simple preconcentration and determination of heavy metal ions using methodology based on DMSPE and TXRF measurement. The suspension of GO-SH was injected into an analyzed water sample; after filtration, the GO-SH nanosheets with adsorbed metal ions were redispersed in a small volume of internal standard solution and deposited onto a quartz reflector. The high enrichment factor of 150 allows obtaining detection limits of 0.11, 0.078, 0.079, 0.064, 0.054, and 0.083 ng mL(-1) for Co(II), Ni(II), Cu(II), As(III), Cd(II), and Pb(II), respectively. Such low detection limits can be obtained using a benchtop TXRF system without cooling media and gas consumption. The method is suitable for the analysis of water, including high salinity samples difficult to analyze using other spectroscopy techniques. Moreover, GO-SH can be applied to the arsenic speciation due to its selectivity toward arsenite.

Measurement of the Direct Oral Anticoagulants Apixaban, Dabigatran, Edoxaban, and Rivaroxaban in Human Plasma Using Turbulent Flow Liquid Chromatography With High-Resolution Mass Spectrometry

Direct oral anticoagulants (DOACs) are prescribed for systemic anticoagulation. Fixed doses are recommended, but dose individualization may be warranted. Functional coagulation assays may be available, but their use requires knowledge of the drug taken. To provide alternative methodology for guiding dosage, we have developed and validated a liquid chromatography-mass spectrometric assay for apixaban, dabigatran, edoxaban, and rivaroxaban at the concentrations attained during therapy. Samples, calibrators, and internal quality controls (100 μL) were mixed with internal standard solution (50 μg/L both dabigatran-13C6 and rivaroxaban-13C6 in acetonitrile) and, after centrifugation (16,400g, 4 minutes), supernatant (100 μL) was injected onto a Cyclone-C18-P-XL TurboFlow column. Analytes were focused onto an Accucore PhenylHexyl (2.1 × 100 mm, 2.6 μm) analytical column and eluted using a methanol + acetonitrile (1 + 1):aqueous ammonium acetate (10 mmol/L) gradient. Data were acquired using high-resolution mass spectrometry in full-scan mode (100-2000 m/z) with data-dependent fragmentation to confirm peak identity. Calibration was linear (1-500 μg/L all analytes). Total analysis time was 6 minutes. Intra-assay imprecision (% RSD) at 1 μg/L was 2.6%, 4.2%, 17.3%, and 9.5% for apixaban, dabigatran, edoxaban, and rivaroxaban, respectively. Mean recovery was 96%-101%. No signal suppression or enhancement was observed. Apixaban, dabigatran, and rivaroxaban were stable over 3 freeze-thaw cycles, after storage at room temperature, and at 2-8°C for up to 2 weeks. Edoxaban was stable over 3 freeze-thaw cycles but showed a mean deterioration of 16% if stored at 2-8°C (2 weeks) and of 18% and 70% (1 day and 2 weeks, respectively) at room temperature. The method is suitable for high-throughput therapeutic drug monitoring of DOACs. The acquisition of full scan data allows for the retrospective identification of metabolites. The method can be used to identify a particular DOAC if information on the drug taken is lacking.

A Direct Method for the Measurement of Everolimus and Sirolimus in Whole Blood by LC–MS/MS Using an Isotopic Everolimus Internal Standard

Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. Whole-blood samples (20 μL) were treated with ammonium bicarbonate (20 μL), zinc sulfate (20 μL), and internal standard solution ((13)C(2)D(4)-everolimus in acetonitrile, 100 μL). After centrifugation, 20 μL of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1-49 μg/L for sirolimus, 1-41 μg/L for everolimus), and a lower limit of quantitation of 1 μg/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%-117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

Dried blood spot testing: a novel approach for the therapeutic drug monitoring of ziprasidone-treated patients.

A novel analytical approach, based on dried blood spot (DBS) testing, has been developed, validated and applied for the first time to the analysis of ziprasidone (ZPR) for the therapeutic drug monitoring (TDM) of schizophrenic patients. DBS represent a more feasible but reliable matrix, alternative to blood and plasma. The assays were carried out using an HPLC method with native fluorescence. Blood drops were applied to DBS cards and dried by microwaves, an internal standard solution was added to the DBS and 5-mm punches were cut out for analysis. ZPR was extracted from DBS with methanol, giving good extraction yields, precision and selectivity results. The method was applied with satisfactory results to DBS samples from psychiatric patients to determine ZPR levels for therapy optimization. This innovative methodology provides reliable and significant TDM information, with important advantages over classical blood sampling in terms of collection, storage and processing.

P12 Determination of UR 144, XLR-11 and their metabolites in hair by LC/MS/MS

Introduction Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on desposition of synthetic cannabinoids including cyclopropylindoles (UR-144 and XLR-11) and their metabolites in hair. UR-144 and XLR-11 have been widely abused and controlled in South Korea recently. Identification of metabolites in hair can be an important proof of synthetic cannabinoids use because it can exclude the possibility of passive smoke exposure. In this study, we described a quantitative method of UR-144 and XLR-11 and their metabolites(UR-144 N -5- hydroxypentyl, UR-144 N -pentanoic acid and XLR-11 4-hydroxypentyl) in hair by liquid chromatography with ESI-MS/MS. Methods The target drugs were extracted in hair by alkaline hydrolysis followed by liquid-liquid extraction(LLE) and solid phase extraction(SPE). After hydrolysis, 1 mL of 0.1 M sodium acetate buffer (pH 4.5) and 0.2 mL of acetic acid were added into hair samples and the samples were extracted with 2 mL of n-hexane:ethyl acetate (9:1). The organic extract was transferred into a disposable test tube and evaporated to dryness at 45 °C under a gentle stream of nitrogen. The extract was reconstituted with 2 ml acetate buffer and SPE was performed using automatic SPE equipment, using hydrophobic and cation exchange SPE column. The extract was evaporated, filtered and analyzed by UHPLC-MS-MS with electrospray ion source in positive-ionization mode. JWH-018-d 9 and JWH-018 N -(5-hydroxypentyl) metabolite-d 5 (50 mL, 100 pg/mL internal standard mixed solution) were used as internal standards for the parent drugs (UR-144 and XLR-11) and their metabolites, respectively. Results Chromatographic separation was completed within 15 min. No interferences were detected in 10 blank hair samples. In intra- and inter-assay precision and accuracy study, CV (%) and bias (%) were below 10. The limit of detection was 0.1 to 2 pg/mg and the limit of quantification was 0.2 to 2 pg/mg, respectively. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration range. No significant variation was observed by different sources of matrices. Conclusion This method was applied to hair samples from 17 individual suspects of XLR-11 or UR-144 use. The range of concentration of XLR-11 and UR-144 were 267.8 ~ 5618.8 pg/mg and 0 ~ 7.87 pg/mg, respectively. The range of concentration of UR- 144 N -5-hydroxypentyl and UR-144 N -pentanoic acid were 1.2 ~ 299.5 pg/mg and 0.5 ~ 127.8 pg/mg, respectively. The range of concentration of XLR-11 4-hydroxypentyl was 0 ~ 11.21 pg/mg. UR- 144 N -4-hydroxypentyl was not detected in all cases. We can assume that UR-144 N -5-hydroxypentyl and UR-144 N -pentanoic acid are the major detectable compounds in hair in case of XLR-11 intake.

Quantitative determination of enzalutamide, an anti‐prostate cancer drug, in rat plasma using liquid chromatography–tandem mass spectrometry, and its application to a pharmacokinetic study

This report details a method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC-MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 µm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple-reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001-1 µg/mL, as well as an intra- and inter-assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 μL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats.

Study of Sample Preparation Method Using Internal Standard Solution to Accurate Quantitative Analysis with &lt;sup&gt;1&lt;/sup&gt;H NMR Spectroscopy

Study of Sample Preparation Method Using Internal Standard Solution to Accurate Quantitative Analysis with &lt;sup&gt;1&lt;/sup&gt;H NMR Spectroscopy

Immunosuppressant therapeutic drug monitoring by LC-MS/MS: Workflow optimization through automated processing of whole blood samples

ObjectivesAlthough, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. MethodsSix-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. ResultsCalibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h. ConclusionsOur findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods.

Simultaneous determination of melamine and cyanuric acid residues in water and urine by solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry method

We aimed to establish a sensitive quantified method for the simultaneous determination of melamine and cyanuric acid residues in water and urine by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry (HILIC-ESI-MS/MS) with the pretreatment of hydrophilic functional silica gel and cation exchange resin mixed solid phase extraction column(MCT), and to investigate the melamine and cyanuric acid residues in 501 water and 216 urine from several province and city. About 100 ml water (or 10 ml urine) was adjusted to pH 3.0 with concentrated hydrochloric acid, and then mixed with the internal standard solution((15)N3-melamine and (15)N3-(13)C3 -cyanuric acid) and 100 ml acetonitrile (10 ml for urine). The solution was cleaned with MCT solid-phase extraction column, and eluted once by 3 ml methanol and twice by 2.5 ml methanol (containing 5% ammonia water). The effluent was collected and dried by N2 flow at 40 °C, and then diluted to 2 mmol/L ammonium acetate containing 90% volume fraction acetonitrile. The completely dissolved solution was then filtered with 0.22 µm organic membrane; and the filtrate was detected by high performance liquid chromatography-tandem mass spectrometry and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. Then we detected the melamine and cyanuric acid residues in 501 water and 216 urine samples collected from several province and city. By the quantification of internal standard (15)N3-melamine and (15)N3-(13)C3-cyanuric acid, the melamine and cyanuric acid were linear in the range of 2.0-1000.0 µg/L with correlation coefficient of 0.9998 and 0.9997. The detection limits of the method were separately 0.4 ng/L (melamine) and 0.3 ng/L (cyanuric acid) for water, and 4.0 ng/L (melamine) and 3.0 ng/L (cyanuric acid) for urine. The average recovery rate was around 95.3%-100.1% with the relative standard deviation (RSD) was <4.02%. Out of the 501 water samples, melamine was detected out in 19.9% (100/501) and cyanuric acid was detected out in 5.2% (26/501). The content was around 0.03-5.00 g/L. Melamine or cyanuric acid was detected out in 24.5% of the urine samples (53/216), with the content around 0.01-1.00 g/L. The established method of solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry can satisfy the requirement for detection of melamine and cyanuric acid residues in all sorts of water and urine. Meanwhile, the two substances widely existed in water and Chinese population.

Enabling Quantitative Analysis in Ambient Ionization Mass Spectrometry: Internal Standard Coated Capillary Samplers

We describe a sampling method using glass capillaries for quantitative analysis of trace analytes in small volumes of complex mixtures (~1 μL) using ambient ionization mass spectrometry. The internal surface of a sampling glass capillary was coated with internal standard then used to draw liquid sample and so transfer both the analyte and internal standard in a single fixed volume onto a substrate for analysis. The internal standard was automatically mixed into the sample during this process and the volumes of the internal standard solution and sample are both fixed by the capillary volume. Precision in quantitation is insensitive to variations in length of the capillary, making the preparation of the sampling capillary simple and providing a robust sampling protocol. Significant improvements in quantitation accuracy were obtained for analysis of 1 μL samples using various ambient ionization methods.

Plasma oxylipin profiling identifies polyunsaturated vicinal diols as responsive to arachidonic acid and docosahexaenoic acid intake in growing piglets

The dose-responsiveness of plasma oxylipins to incremental dietary intake of arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) was determined in piglets. Piglets randomly received one of six formulas (n = 8 per group) from days 3 to 27 postnatally. Diets contained incremental ARA or incremental DHA levels as follows (% fatty acid, ARA/DHA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D1) 0.66/0.33; and (D2) 0.67/0.62, resulting in incremental intake (g/kg BW/day) of ARA: 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 at constant DHA intake (0.82 ± 0.05), or incremental intake of DHA: 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 at constant ARA intake (0.54 ± 0.04). Plasma oxylipin concentrations and free plasma PUFA levels were determined at day 28 using LC-MS/MS. Incremental dietary ARA intake dose-dependently increased plasma ARA levels. In parallel, ARA intake dose-dependently increased ARA-derived diols 5,6- and 14,15-dihydroxyeicosatrienoic acid (DiHETrE) and linoleic acid-derived 12,13-dihydroxyoctadecenoic acid (DiHOME), downstream metabolites of cytochrome P450 expoxygenase (CYP). The ARA epoxide products from CYP are important in vascular homeostatic maintenance. Incremental DHA intake increased plasma DHA and most markedly raised the eicosapentaenoic acid (EPA) metabolite 17,18-dihydroxyeicosatetraenoic acid (DiHETE) and the DHA metabolite 19,20-dihydroxydocosapentaenoic acid (DiHDPE). In conclusion, increasing ARA and DHA intake dose-dependently influenced endogenous n-6 and n-3 oxylipin plasma concentrations in growing piglets, although the biological relevance of these findings remains to be determined.

Determination of epichlorohydrin in drinking water by isotope dilution gas chromatography-mass spectrometry

To establish a method for determination of the epichlorohydrin in drinking water by isotope dilution gas chromatography-mass spectrometry (GC-MS). The internal standard solution D5-epichlorohydrin was added in drinking water sample. The epichlorohydrin was firstly collected by active carbon, and the adsorbent was then centrifuged at 2739 × g for 10 min to remove water. Finally, the epichlorohydrin was desorbed by dipping the active carbon in 1.0 ml acetone for 1 h. The desorbed solution was tested by GC-MS and quantified with isotopic internal standards. The detection limit, precision and accuracy of the assay were evaluated. This method was adopted to detect the epichlorohydrin in drinking water for 25 batches in a city. The determination method of epichlorohydrin represented a good linear relationship in the range of 0.0645-3.8700 µg/L, the linear regression equation was Y = 2.828X + 4.91 × 10(-2) (r > 0.999). When the epichlorohydrin concentration were 0.0806, 0.3230 and 3.2300 µg/L, the relative standard deviations (RSD) were 7.9%, 4.7% and 3.1%, respectively. The average recoveries were from 95.7% to 98.7%. The limit of detection (LOD) was 0.015 µg/L, limit of quantification (LOQ) was 0.052 µg/L. The content of epichlorohydrin in the 25 cases of drinking water was under the limit of detection. The method is more simple than the national standard method, with high sensitivity, accuracy and good reproducibility, which is suitable for detection of the trace amounts of epichlorohydrin in drinking water.

Quantitative analysis of morphine in dried blood spots by using morphine-d3 pre-impregnated dried blood spot cards

Two different internal standard dried blood spot (DBS) pre-impregnation procedures (prior to blood spotting) were investigated. In the first procedure DBS pre-impregnation is performed by immersing the DBS card fully into an internal standard solution. In the second procedure pre-impregnation is performed by pipetting a certain volume of an internal standard solution onto the DBS card. Morphine-d3 was used as the model compound for all experiments. The pre-impregnation procedure by immersing was further investigated with respect to homogeneity of impregnation, influence of different blood spotting techniques and the influence of spotting different blood volumes on the internal standard distribution, calibration and stability of pre-impregnated cards. Finally, the immersing procedure was used for the analysis of morphine in dried blood spots and the results were compared to the conventional procedure in which the internal standard morphine-d3 was added to the extraction solvent. The new pre-impregnated cards couple simplicity of operation and convenient use in the field to results equivalent to the conventional procedure.

Internal Standard Application to Dried Blood Spots by Spraying: Investigation of The Internal Standard Distribution

The scientifically and logistically best way of application of the internal standard (IS) in the analysis of dried blood spots (DBS) analysis is still a matter of debate and investigation. Most commonly the IS is added in the solvent used for extraction of the discs punched from DBS. In this case, the recovery of the non-extracted IS is complete while the recovery of the analyte extracted from DBS is different from the IS. An alternative way for addition of the IS was investigated. A homogeneous distribution and absorption of the test compound across the spots was demonstrated by spraying a solution of a radiolabeled test compound (mimicking an IS solution) onto DBS. This spray-on technique is convenient and easily automatable. Spraying of the solution was rapid, precise and reproducible, and therefore seems to be suitable for routine analysis of DBS by offline and online extraction.