High-throughput On-line Solid-phase Research Articles

Caffeine quantification in dietary supplements using high-throughput on-line solid phase extraction coupled to Venturi easy ambient sonic-spray ionization mass spectrometry.

Caffeine is present in a large number of beverages and is an additive used in dietary supplements. Therefore, the concern about its quality and safety for consumers has been increasing and hence requires faster and simpler analytical methods to determine the caffeine amount. The high-throughput analysis is an appropriate solution to pharmaceuticals, bioanalysis, forensic and food laboratory routines. In this sense, Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS), a specific ambient ionization source, is suitable to enable direct analysis of sample solutions in real time and is appropriate to be coupled to liquid chromatography (LC). The development of an on-line solid phase extraction system coupled to V-EASI-MS optimizes the advantages of LC-MS hyphenation by enhancing the figures of merit of the analytical method according to AOAC guidelines and simultaneously minimizing the runtime analysis to 1.5 min per sample, as well as sample preparation steps and solvent consumption, which is currently a challenge for quantitative applications of ambient ionization MS.

B-310 High Through-put Online Solid Phase Extraction of Immunosuppressive Drugs From Whole Blood on Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) in a Tertiary Hospital Laboratory

Abstract Background Cyclosporine A and Tacrolimus are immunosuppressive drugs that are commonly used in solid organ transplant and are characterized by a relatively narrow therapeutic index, potential drug interactions and significant intra- and inter-individual variability. Therapeutic drug monitoring is therefore beneficial for optimizing dosage regimens, preventing rejection of the graft and reducing toxicities in transplant recipients receiving immunosuppressive drug therapy. A commercial assay (RECIPE ClinMass LC-MS/MS Advanced kit) for simultaneous measurement of immunosuppressants in whole blood using LC-MS/MS methodology has been adapted for assay performance evaluation in our laboratory on the Agilent 1290 Infinity II liquid chromatograph coupled to an Agilent 6470 triple quadrupole mass spectrometer. Our study evaluated the analytical performance of Cyclosporine A and Tacrolimus on the RECIPE assay and compared it against our existing in-house LC-MS/MS assay. Methods The RECIPE ClinMass® LC-MS/MS Advanced Immunosuppressants assay kit consist of mobile phase solvents, precipitation reagents, stable isotopic labeled internal standards, solid phase extraction and analytical columns, lyohilized whole blood calibrators and quality controls. Drug analytes were extracted from whole blood using single-step protein precipitation followed by on-line solid phase extraction on the LC, eluted on isocratic chromatography and detected on positive ESI MS ionisation mode. The RECIPE assay run time is under 3 min. Performance validation parameters included within/total assay imprecision, linearity, recovery, limits of detection (LOD)/quantification (LOQ) and carry-over. Method correlation studies using fresh patient whole blood specimens (n = 212 Cyclosporine A; n = 120 Tacrolimus) were performed on existing in-house assay on Agilent 6460 LC-MS/MS and the RECIPE assay on Agilent 6470 LC-MS/MS systems. Results Within run and total imprecision for Cyclosporine A and Tacrolimus on the RECIPE assay were determined to be ≤4.4% and ≤7.5% on the Agilent 6470 LC-MS/MS. The assay demonstrated linearity across the analytical measurement range up to 1500 ug/L for Cyclosporine A and up to 55 ug/L for Tacrolimus. Analytical recoveries obtained for both drugs ranged between 93%–100%. The LODs were assessed to be 13.0 ug/L and 0.70 ug/L for Cyclosporine A and Tacrolimus respectively. LOQs were assessed to be 22.7 ug/L and 1.30 ug/L for Cyclosporine A and Tacrolimus respectively. Results of carry-over studies were insignificant. Participation in the CAP Immunosuppressive Drugs External Quality Assessment Survey showed good agreement with LC-MS/MS peers. Method correlation with in-house assay demonstrated good agreement: (1) Cyclosporine A: Passing Bablok regression RECIPE = 0.94 (In-house) + 17.2, Bland Altman mean bias of +2.0%, Spearman correlation coefficient of 0.985; (2) Tacrolimus: Passing Bablok regression of RECIPE = 1.03 (In-house) - 0.2, Bland Altman mean bias of -0.2%, Spearman correlation coefficient of 0.988 Conclusion The RECIPE ClinMass® LC-MS/MS Immunosuppressants assay performed within manufacturer’s specifications except for LOD/LOQs which were determined at higher concentrations. The assay is adaptable to contemporary LC-MS/MS systems and confer benefits from rapid turnaround time (<3 min), simplified specimen preparation procedures and ready prepared reagents. The advantages allow enhancements in workflow efficiency and ease of staff training in our laboratory and the assay is suitable for routine clinical use in high workload clinical laboratories.

High throughput online solid-phase extraction tandem mass spectrometry for immunosuppressive drugs determination in whole blood

High throughput mass spectrometry-based clinical assay technologies have emerged in response to the increase in the number of patients who require fast, accurate, and robust analysis methods. Typical runtimes for quantitation based on liquid chromatography – tandem mass spectrometry (LC–MS/MS) are in the order of a few minutes per sample. The use of tandem mass spectrometry with online solid phase extraction (SPE-MS/MS) instead of chromatography columns can offer high-throughput determination without compromising sensitivity, precision, and accuracy. In this communication, we describe a newly developed fast, accurate, and robust method on the Agilent RapidFire-MS/MS System (RF-MS/MS) for the simultaneous determination of Sirolimus, Everolimus, Tacrolimus and Cyclosporine in whole blood. The commercial multilevel calibrator and quality control (from Chromsystems) were precipitated using a zinc sulfate and methanol mixture solution containing the deuterated internal standards. Precipitated samples were centrifuged and transferred to a 96-well plate for analysis. Immunosuppressive drugs analysis was performed online with C18 extraction small cartridge onto an Agilent RapidFire 400 and 6495 Triple Quadrupole Mass Spectrometer by MRM mode in positive electrospray ionization. Imprecision and accuracy were investigated analyzing 10 Asqualab External Quality Controls. We compared the results obtained with both techniques. The new method showed good linearity 2.35–24.90 μg/L for Sirolimus, 2–21.80 μg/L for Everolimus, 1.43–23.20 μg/L for Tacrolimus and 22–757 μg/L for Cyclosporine with an r 2 > 0.995. RF-MS/MS showed good intraday and interday precision (< 10%) for all immunosuppressive drugs. High accuracy was achieved with 10 samples from the Asqualab program (< 10%). No significant carryover was measured for any of the drug analytes and high endurance of the solid phase extraction cartridge (> 15,000 injections). The RapidFire – MS/MS system provided excellent agreement with our routine LC–MS/MS method. Its main advantage is the high throughput analysis of 13 s per sample, the low consumption of toxic organic solvents, and the high robustness. It could represent an alternative to LC–MS/MS instruments in therapeutic drug monitoring in human biological fluids.

Per- and polyfluoroalkyl substances in serum and associations with food consumption and use of personal care products in the Norwegian biomonitoring study from the EU project EuroMix

BackgroundHuman exposure to chemicals through the oral, dermal, or inhalation routes is significant. To assess this exposure, a human biomonitoring study was conducted in Norway to examine the plausibility of source-to-dose calculations for chemical mixtures. Per- and polyfluoroalkyl substances (PFASs) are man-made compounds used for their surfactant properties, and several are persistent and bioaccumulative. Some PFASs are toxic and are regarded as endocrine disruptors and have been shown to suppress immune function and affect cholesterol homeostasis. Using the participants from the EuroMix BM study, we set out to describe PFAS concentrations and to evaluate associations with diet and use of personal care products (PCPs). MethodsParticipants (44 males and 100 females) kept detailed diaries on their food consumption and their PCP use for two non-consecutive days. All urine (24 h) and blood samples were collected at the end of each study day. Levels of 25 PFASs were analysed in serum from study day 1 using a high throughput online solid phase extraction ultra-high-performance liquid chromatography tandem mass spectrometry method. Multivariable linear regressions were performed between each food and PCP category and each chemical and were sex-stratified when the consumption of food or use of PCPs was significantly different between men and women. ResultsEight PFASs were detected in all analysed samples (PFHxS, PFHpS, PFOS, PFOA, PFNA, PFDA, PFUnDA and PFDoDA), and four PFASs were below the limit of detection (PFOPA, PFDPA, PFHxA, and EtFOSA). Several PFASs were found to be positively associated with fish consumption (PFOS, PFNA, PFUnDA, PFDoDA, PFDA, PFDS and PFTrDA). Sunscreen, mouthwash, and lip gloss/lip balm were found to be positively associated with PFASs (PFOA, PFTrDA, and PFOSA). ConclusionThe participants in the EuroMix study were exposed to PFASs through their diet and PCP use. Several foods and PCPs were found to be potential sources of exposure to PFASs.

High throughput online solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry method for polyfluoroalkyl phosphate esters, perfluoroalkyl phosphonates, and other perfluoroalkyl substances in human serum, plasma, and whole blood

A rapid, sensitive and reliable method was developed for the determination of a broad range of poly- and perfluoroalkyl substances (PFASs) in various blood matrices (serum, plasma, and whole blood), and uses only 50 μL of sample material. The method consists of a rapid protein precipitation by methanol followed by high throughput online solid phase extraction (SPE), ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), and negative electrospray ionization detection. The method was developed for simultaneous determination of twenty-five PFASs, including polyfluoroalkyl phosphate esters (PAPs; 6:2, 8:2, 6:2/6:2, and 8:2/8:2), perfluoroalkyl phosphonates (PFPAs; C6, C8, and C10), perfluoroalkyl sulfonates (PFSAs; C4, C6, C7, C8, and C10), perfluoroalkyl carboxylates (PFCAs; C5C14), and perfluoroalkyl sulfonamides (FOSAs; C8, N-methyl, and N-ethyl). High linearity of matrix-matched calibration standards (correlation coefficients, R = 0.99–0.999) were obtained in the range of 0.006–45 ng mL−1 blood. Excellent sensitivity was achieved with method detection limits (MDLs) between 0.0018 and 0.09 ng mL−1, depending on the compound and matrix. The method was validated for serum, plasma, and whole blood (n = 5 + 5) at six levels in the range 0.0180–30 ng mL−1. The accuracy (n = 5) was on average 102± 12%. The intermediate precision (n = 10) ranged from 2 to 40% with an average between-batch of analyses difference of 10± 10%. Two human serum samples from a former interlaboratory comparison were analyzed and the differences between the applied method and the consensus values were below ≤22% (n = 5). The method was also successfully applied to samples of human plasma and whole blood with coefficients of variation in the range 0.8–15.2% (n = 5).

Development and validation of a high-throughput online solid phase extraction – Liquid chromatography – Tandem mass spectrometry method for the detection of tetrodotoxin in human urine

Tetrodotoxin (TTX) is an extremely potent paralytic toxin responsible for yearly illness and death around the world. A clinical measurement is necessary to confirm exposure because symptoms of TTX intoxication cannot be distinguished from other paralytic toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of TTX in human urine with tandem mass spectrometry. The reportable range for the method was 2.80 - 249 ng/mL in urine with precision and accuracy within 15% as determined for all quality control samples. No isotopically-labeled internal standard is available for TTX; thus a surrogate internal standard, voglibose, was investigated to compensate for matrix effects and ionization suppression. However, upon evaluation, voglibose was ineffective for this purpose. This new online method rapidly identifies TTX, facilitating the work of public health authorities and providing support to monitoring programs worldwide.

High-throughput online solid-phase extraction tandem mass spectrometry: Is it right for your clinical laboratory?

With increasing patient volumes and growing demands for rapid turnaround on many clinical tests, there is a demand for high-throughput technologies. High-Throughput Online Solid-Phase Extraction Tandem Mass Spectrometry is an example of one technology that can achieve these desired results and the RapidFire 365 Mass Spectrometry system from Agilent Technologies is one vendor's solution. The key advantage of the RapidFire system is its speed of analysis and throughput. While the Agilent RapidFire system cannot be utilized for every clinical analyte, it does work well for several classes of medications including immunosuppressants, anticonvulsants and antineoplastic agents where dosage adjustments are made to maximize efficacy and minimize toxicity once results are available. In the end, high throughput tandem mass spectrometry has numerous benefits and limitations which must be weighed for each clinical analyte to determine if it's the right solution for your lab. This article will specifically discuss the Agilent RapidFire system.

Fast determination of hydroxylated polychlorinated biphenyls in human plasma by online solid phase extraction coupled to liquid chromatography-tandem mass spectrometry

Hydroxylated polychlorinated biphenyls (OH-PCBs) have been shown to be strongly retained in human blood causing endocrine-related toxicity, particularly on the thyroid system. Traditionally, analytical methods for the determination of OH-PCBs require labor-intensive and long-time consuming sample preparation with several extraction, evaporation and cleanup procedures steps and, in some cases, derivatization prior to the analysis by gas or liquid chromatography-mass spectrometry (GC–MS or LC-MS). The present study developed and validated a novel, sensitive and high throughput online solid phase extraction (SPE) method coupled to LC-tandem mass spectrometry (MS/MS) for the separation and quantitation of relevant congeners of OH-PCBs in human plasma. The developed method presented limits of quantification (LOQ) ranging from 0.02 to 0.5 ng mL−1 and extraction recoveries from 71 to 134% for all congeners, requiring small amount of sample (only 100 μL) and minimal sample preparation. In order to evaluate the applicability of the method, preliminary tests (N = 93) were conducted in plasma from individuals occupationally exposed to very high levels of PCBs in a German cohort. Penta-through hepta-chlorinated OH-PCBs were the predominant congeners in human plasma with concentrations up to 44.5 ng mL−1, while lower chlorinated OH-PCBs were occasionally detected. In addition, a new PCB 28 metabolite has been synthesized and identified for the first time in human plasma and associations between OH-PCBs and their parent compounds in the studied cohort were also assessed.

Evaluation of a High-Throughput Online Solid Phase Extraction–Tandem Mass Spectrometry System for In Vivo Bioanalytical Studies

High throughput-solid phase extraction tandem mass spectrometry (HT-SPE/MS) is a fully automated system that integrates sample preparation using ultrafast online solid phase extraction (SPE) with mass spectrometry detection. HT-SPE/MS is capable of conducting analysis at a speed of 5-10 s per sample, which is several fold faster than chromatographically based liquid chromatography-mass spectrometry (LC-MS). Its existing applications mostly involve in vitro studies such as high-throughput therapeutic target screening, CYP450 inhibition, and transporter evaluations. In the current work, the feasibility of utilizing HT-SPE/MS for analysis of in vivo preclinical and clinical samples was evaluated for the first time. Critical bioanalytical parameters, such as ionization suppression and carry-over, were systematically investigated for structurally diverse compounds using generic SPE operating conditions. Quantitation data obtained from HT-SPE/MS was compared with those from LC-MS analysis to evaluate its performance. Ionization suppression was prevalent for the test compounds, but it could be effectively managed by using a stable isotope labeled internal standard (IS). A structural analogue IS also generated data comparable to the LC-MS system for a test compound, indicating matrix effects were also compensated for to some extent. Carry-over was found to be minimal for some compounds and variable for others and could generally be overcome by inserting matrix blanks without sacrificing assay efficiency due to the ultrafast analysis speed. Quantitation data for test compounds obtained from HT-SPE/MS were found to correlate well with those from conventional LC-MS. Comparable accuracy, precision, linearity, and sensitivity were achieved with analysis speeds 20-30-fold higher. The presence of a stable metabolite in the samples showed no impact on parent quantitation for a test compound. In comparison, labile metabolites could potentially cause overestimation of the parent concentration if the ion source conditions are not optimized to minimize in-source breakdown. However, with the use of conditions that minimized in-source conversion, accurate measurement of the parent was achieved. Overall, HT-SPE/MS exhibited significant potential for high-throughput in vivo bioanalysis.

Development of a High-Throughput Online Solid-Phase Extraction/Tandem Mass Spectrometry Method for Cytochrome P450 Inhibition Screening

A high-throughput online solid-phase extraction/tandem mass spectrometry (online SPE/MS/MS) system has been developed to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of 8 test concentrations and against a panel of 6 cytochrome P450 (CYP) enzymes, 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. Previously, a postassay pooling and a 2-min gradient LC/MS/MS method had been reported to increase sample throughput, allowing for a 96-well plate of samples to be analyzed in under 4 h. The development of a new online SPE/MS/MS system has reduced the analysis time to less than 15 min per 96-well plate, translating to a 15-fold time savings compared to the 2-min LC/MS/MS method. Sampling precision without internal standard correction ranged from 3.1% to 5.6% relative standard deviation, and the carryover was determined to be between 1.0% and 4.1%. One hundred twenty in-house compounds were assayed and pooled for analyses using both the online SPE/MS/MS and LC/MS/MS, and the correlation coefficients ranged from 0.89 to 1.13, when comparing the IC(50) results obtained from the 2 approaches for each of the CYP enzymes.

Plasma tryptophan, kynurenine and 3-hydroxykynurenine measurement using automated on-line solid-phase extraction HPLC–tandem mass spectrometry

Tryptophan metabolism plays a key role in several (patho)physiological conditions. In order to study the clinical importance of tryptophan and its predominant metabolites (kynurenines), it is important to be able to measure large series of samples with high accuracy and reliability. We aimed to develop a high-throughput on-line solid-phase extraction-liquid chromatographic–tandem mass spectrometric (XLC–MS/MS) method that enables the measurement of tryptophan and its metabolites kynurenine and 3-hydroxykynurenine in plasma. Fifty microliters plasma equivalent was pre-purified by automated on-line solid-phase extraction, using strong cation exchange (PRS, propylsulphonic) cartridges. Chromatographic separation of the analytes and deuterated analogues occurred by C18 reversed phase chromatography. Mass spectrometric detection was performed in the multiple reaction-monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Total run-time including sample clean-up was 8 min. Intra- and inter-assay analytical variations were less than 9%. Linearity in the 0.11–1200 (tryptophan) and 0.050 and 0.023–45 μmol/L (kynurenine and 3-hydroxykynurenine, respectively) calibration range was excellent ( R > 0.99). Detection limits were 30 nmol/L for tryptophan, 1 nmol/L for kynurenine and 5 nmol/L for 3-hydroxykynurenine. Reference intervals for 120 healthy adults were 45.5–83.1 μmol/L (tryptophan), 1.14–3.02 μmol/L (kynurenine), <0.13 μmol/L (3-hydroxykynurenine) and 19.0–49.8 for tryptophan-to-kynurenine ratio. Blood sampling for tryptophan and tryptophan-to-kynurenine ratio should be performed before breakfast, due to biological variation during the day. This study describes how plasma tryptophan, kynurenine and 3-hydroxykynurenine can be measured accurately and precisely by automated high-throughput XLC–MS/MS.

High-throughput on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of 14 antidepressants and their metabolites in plasma

A rapid, sensitive and fully automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma. Integration of the sample extraction and LC separation into a single system permitted direct injection of the plasma without prior sample pre-treatment. The applied gradient ensured the elution of all the examined drugs within 14 min and produced chromatographic peaks of acceptable symmetry. The total process time was 20 min and only 50 μL of plasma was required. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated compounds. The use of SPE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Extraction was found to be both reproducible and efficient with recoveries >99% for all the analytes. The method showed excellent intra-assay and inter-assay precision (relative standard deviation (RSD) and bias <20%) for quality control (QC) samples spiked at a concentration of 40, 200 and 800 μg/L and the r 2 > 0.99 over the range investigated (10–1000 μg/L). Limits of quantification (LOQs) were estimated to be 10 μg/L. Furthermore, the processed samples were demonstrated to be stable for at least 48 h, except for clomipramine and norclomipramine, where a slight negative trend was observed, but did not compromise the quantification. The method was subsequently applied to authentic samples previously screened by a routine HPLC method with diode array detection (DAD).

High throughput on-line solid phase extraction/tandem mass spectrometric determination of paclitaxel in human serum.

The feasibility of high throughput on-line solid phase extraction/tandem mass spectrometry (SPE/MS/MS) is tested for target analysis of paclitaxel in human serum. The use of a dual Prospekt system, with parallel SPE and elution directly to the mass spectrometer, resulted in a cycle time of 80 seconds for the entire, fully automated assay. The assay proved to be linear from 1 to 1000 ng/mL. Cartridges packed with small sorbent particles functioned both as SPE cartridges and as short analytical columns.