Automation of chromatographic peak review and order to result data transfer in a clinical mass spectrometry laboratory.

INTRODUCTION: Mass spectrometry-based assays have increasingly been implemented in clinical laboratories for their multiplexing capacity and high specificity and sensitivity. However, these methods are often associated with labor-intensive and error-prone data-related workflows, due to the volume of data generated that is often manually reviewed and resulted. We aimed to establish a system within our clinical mass spectrometry laboratory to facilitate data 'flow' from electronic medical record order to result and to automate processes for chromatogram peak review. The processes and validation are described for a 25-hydroxyvitamin D assay. METHODS: Automating chromatogram review and order to result data transfer required flat file interfacing, file transfers of standardized data formats, barcode scanning, and software for peak processing and review. Validation of the automated workflow involved (1) correlation of quantified results generated by two chromatogram analysis methods: Waters TargetLynx and Indigo Bioautomation ASCENT, (2) manual verification of quality assurance flags applied in ASCENT, and (3) testing data flow and integrity across all the systems from order to result. Efficiency and quality improvements were assessed through calculation of batch review times and rates for autoverification and manual manipulations. RESULTS: The correlation of TargetLynx and ASCENT quantitation methods for 25-hydroxyvitamin D2 in patient samples yielded slope of 0.99 (95% CI: 0.989 to 0.996), intercept of 0.46 (95% CI: 0.363 to 0.565), with r = 0.999. The correlation for the D3 fraction showed Deming regression slope of 0.98 (95% CI: 0.969 to 0.989), intercept of 0.06 (95% CI: -0.115 to 0.313), and r = 0.995. Results from both quantitation approaches were also compared to the assigned value in CDC reference samples. The mean bias relative to the CDC was 4.6% for ASCENT and 2.5% for TargetLynx. The median time for chromatogram review of a full 96-well plate of vitamin D results is reduced from approximately 2 h to 14 min and 80% of batches were reviewed within 30 min. Instead of 100% peak review, technologists review only the peaks that have been flagged by the system based on applied rules. Analysis of full plate batches showed that 2-20% of peaks per batch were flagged for manual review. Manipulations made by technologists during chromatogram review were reduced by 75% when using the automated versus manual system. CONCLUSIONS: We describe a system to facilitate data 'flow' from electronic order to result and to automate chromatogram peak review in a clinical liquid chromatography mass spectrometry assay for 25-hydroxyvitamin D. This eliminated manual result entry, repetitive transcription, and unnecessary review of high quality data while enabling systematic evaluation of data quality indicators. The new processes were accurate, improved the data review and processing times, and helped to reduce manual manipulations during chromatogram review.

Quantification of Arginine and Its Methylated Derivatives in Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

Arginine is the substrate for nitric oxide synthases (NOS), thus the production of nitric oxide (NO) is based on arginine availability. Arginine is methylated through the activity of protein arginine methyltransferases (PRMT1 and PRMT2), to form asymmetrical dimethylarginine (ADMA) and symmetrical dimethylarginine (SDMA). These compounds have gained interest in recent years due to their influence on NO production rates and association with cardiovascular and renal diseases. The accurate and precise measurement of arginine and its methylated derivatives is needed for research studies investigating their role(s) in NO bioavailability and development of disease. We describe a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for quantifying arginine, ADMA, and SDMA requiring only 50 µL of plasma. The sample preparation involves addition of internal standards (ADMA-d7 for ADMA and SDMA, and (13)C6 -arginine for arginine) prior to protein precipitation with LCMS grade acetonitrile. Samples are centrifuged and supernatant is dried under nitrogen gas at 50 °C. Samples are reconstituted with mobile phase (ammonium acetate-formic acid-water). Arginine, ADMA, and SDMA are separated using an isocratic HPLC method on a 3 µM silica analytical column. MS/MS detection is performed in the multiple-reaction monitoring (MRM) mode and the transitions monitored are m/z 203 to m/z 70 for ADMA and SDMA, m/z 210 to m/z 77 for ADMA-d7, m/z 175 to m/z 70 for arginine, and m/z 181 to m/z 74 for (13)C6-arginine.

Quantification of Iohexol in Serum by High-Performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Iohexol is a nonradioactive contrast medium, and its clearance from serum or urine is used to measure glomerular filtration rate (GFR). GFR is the most useful indicator of kidney function and progression of kidney disease. GFR determination using iohexol clearance is increasingly being applied in clinical practice, given its advantages over and correlation with inulin. We describe a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for iohexol clearance, requiring only 50 µL of serum. The sample preparation involves protein precipitation with LC/MS-grade methanol, containing ioversol as the internal standard. Samples are centrifuged and supernatant is dried under nitrogen gas at room temperature. Samples are reconstituted with mobile phase (ammonium acetate-formic acid-water). Iohexol is separated using an HPLC gradient method on a C-8 analytical column. MS/MS detection is in the multiple-reaction monitoring (MRM) mode and the transitions monitored are m/z 822.0 to m/z 804.0 and m/z 807.0 to m/z 588.0 for iohexol and ioversol, respectively.

Determination of iohexol in human serum by a semi-automated liquid chromatography tandem mass spectrometry method.

OBJECTIVES: Measured glomerular filtration rate (mGFR) is the best indicator of renal function in children and adolescents. GFR determination using iohexol clearance has been increasingly accepted and applied in clinical practice because it is accurate, readily available, non-radioactive, safe and is used intravenously even in the presence of renal disease. This study describes the development and evaluation of a semi-automated method for determination of iohexol in human serum using liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Iohexol was extracted from serum using a MICROLAB® NIMBUS4 automation robot and supernatant was dried under nitrogen gas and reconstituted in mobile phase. Ioversol was used as the internal standard. Chromatography was performed using a C-8 analytical column (Phenomenex, 3 µm, 50 × 3.0 mm I.D.) at room temperature and a gradient LC method on a Waters 2795 Alliance HT HPLC system. The flow rate was 0.5 mL/min and the retention times were 2.36 min and 2.14 min for iohexol and ioversol, respectively. Detection by MS/MS was achieved using a (Micromass Quattro Micro) tandem mass spectrometer operated in the ESI-positive mode. The multiple-reaction monitoring (MRM) method used ion transitions m/z 821.9 to 803.7 for iohexol and m/z 807.9 to 588.7 for ioversol. Method validation studies were conducted to determine the linearity, accuracy, precision, matrix effects and stability. A method comparison of blinded, residual patient samples was conducted with a well-established method. RESULTS: The method was linear from 7.7 µg/mL to 2000.0 µg/mL. The low limit of quantification and the detection limit were established at 7.7 and 3.0 µg/mL, respectively. Within-run and between-run precisions were found to be <6% CV and measured values deviated no more than 5% from target concentrations. Carryover and matrix effects were not significant. Comparison to a well-established method showed very good agreement with correlation coefficient of 0.996 for iohexol and 0.993 for GFR/1.73 m(2). CONCLUSIONS: This method accurately and precisely quantifies iohexol in 50 µL of serum, enabling determination of mGFR by iohexol clearance. The method is highly correlated to a reference method. Use of an automated liquid handler reduces labor-intensive, manual sample preparation steps. The stability of this analyte and the robustness of this assay fit well within our clinical workflow and we have successfully applied this method to determine mGFR in pediatric patients.

Measurement of arginine derivatives in pediatric patients with chronic kidney disease using high-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: The arginine derivatives asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) interfere with endothelial nitric oxide synthesis. Plasma ADMA and SDMA have been shown to be risk factors for cardiovascular disease and/or kidney function deterioration in a variety of patient populations. METHODS: We developed a method to quantitatively measure arginine, ADMA, and SDMA using HPLC-tandem mass spectrometry. 13C6-L-Arginine was used as the internal standard, while the derivatives were separated on a silica column in less than 14 min. Plasma levels of ADMA, SDMA, and arginine were measured in children with stage II or III chronic kidney disease (CKD) and age- and gender-matched siblings. RESULTS: The chromatography exhibited no observable ion suppression in the patient specimens tested. There was no apparent carryover for any of the analytes. The assay was linear over 0.32-2.29, 0.23-4.43, and 1.00-303.89 micromol/L for ADMA, SDMA, and arginine, respectively. Plasma ADMA, SDMA, and arginine (mean+/-SD) were 1.10+/-0.35, 2.06+/-1.11, and 57.93+/-22.10 mumol/L for children with CKD, and 0.78+/-0.16, 0.71+/-0.23, and 65.29+/-21.30 micromol/L for the healthy siblings. CONCLUSIONS: The method exhibited no observable ion suppression in the patient specimens tested and has an acceptably short analytical cycle time. Children with CKD had higher levels of ADMA and SDMA than the healthy siblings.

Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry.

BACKGROUND: Sirolimus is widely used as an immunosuppressant, along with calcineurin inhibitors. Because of its variable pharmacokinetics and narrow therapeutic range, therapeutic drug monitoring of sirolimus is critical to optimize its therapeutic effect and to minimize toxicity. Although liquid chromatography/tandem mass spectrometry is considered the method of choice, the technical and financial challenges of this method are obstacles to its use. A microparticle enzyme immunoassay on the Abbott IMx has recently been reintroduced to the clinical diagnostic market. METHODS: We evaluated this immunoassay using high-performance liquid chromatography/tandem mass spectrometry as the reference method. Precision and carryover were evaluated using an expanded CLSI EP10-A2 protocol. Linearity was studied by serial dilution of high-level whole blood samples, and clinical utility was demonstrated by correlation with the reference method using 56 de-identified pediatric patient samples. RESULTS: The total imprecision was less than 12% across the concentrations tested. The method was linear from 2.6 to 31 nM. The immunoassay showed a mean positive bias of 11.5% in patient specimens relative to high-performance liquid chromatography/mass spectrometry (p<0.001), with a correlation coefficient (R) of 0.953. CONCLUSION: We conclude that the reintroduced immunoassay is useful for therapeutic drug monitoring of sirolimus.

Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry.

Low levels of serum testosterone typically found in women and children cannot be reliably measured by immunoassay. We developed a simple and sensitive method using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Sample preparation involved protein precipitation of serum (1.0 mL) with acetonitrile containing the internal standard (testosterone-d3) followed by liquid extraction with methylene chloride. The chromatographic cycle per specimen was 10 min. The performance was evaluated according to the CLSI EP10-A2 protocol. Within- and between-run imprecision was 20.9%, 2.29% and 1.80%, and 1.81%, 3.58% and 2.97% at mean concentrations of 0.17, 14.1 and 28.8 nM, respectively, with no apparent carryover. The method was linear from 0.21 to 53.1 nM and the analytical recovery was 100.5-106.2% across the concentrations tested. There was no interference observed from other steroids that were tested. Correlation using de-identified patient specimens with a commercial HPLC-MS/MS method showed a slope of 0.991, an intercept of -0.017 and a correlation coefficient (R(2)) of 0.998 by linear regression over concentrations ranging from 0.21 to 16.7 nM. In conclusion, we report here an HPLC-MS/MS method suitable for clinical measurement of serum testosterone.

A fast and simple high-performance liquid chromatography/mass spectrometry method for simultaneous measurement of whole blood tacrolimus and sirolimus.

CONTEXT: Combined immunosuppressant therapy using tacrolimus and sirolimus has demonstrable benefits. Simultaneous chromatographic monitoring of whole blood tacrolimus and sirolimus is useful for reducing reagent consumption and turnaround time. We report here a simple and rapid method using high-performance liquid chromatography/mass spectrometry for simultaneous measurement of whole blood tacrolimus and sirolimus. OBJECTIVE: To develop and validate a high-performance liquid chromatography/mass spectrometry method that is suitable for clinical laboratories and that is simple, rapid, and cost-effective. DESIGN: Whole blood (80 microL) was mixed with zinc sulfate solution, followed by protein precipitation with acetonitrile containing the internal standards. After brief centrifugation, the supernatant (20 microL) was injected onto a C18 guard column. The drug and the internal standard ammonium adducts were monitored by multiple reaction monitoring. One-point calibration at levels of 200 ng/mL (249 nM) tacrolimus and 100 ng/mL (109 nM) sirolimus was prepared by adding tacrolimus and sirolimus to immunosuppressant-free whole blood. RESULTS: The assay took 2.5 minutes per sample injection. The total imprecision was between 2.46% and 7.04% for tacrolimus and between 5.22% and 8.30% for sirolimus across the concentrations tested. No carryover was observed, and recoveries were 92% to 98% for tacrolimus and 100% for sirolimus at all levels tested. The tacrolimus was linear from 0.52 to 155.5 ng/mL (0.65-193.4 nM), and sirolimus was linear from 0.47 to 94.8 ng/mL (0.51-103.7 nM). Biases of correlations with commercial methods were within 7%. CONCLUSIONS: This improved method is simple, fast, cost-effective, and suitable for clinical laboratories. It has been implemented for routine clinical monitoring of posttransplantation immunosuppressant therapy.