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Original Article
액체크로마토그래피-질량분석법 기반 전혈 내 면역억제제 약물농도 정량검사에 접목한 Andrew+ 검체 로봇 전처리방법 평가
Evaluation of a Robotic Liquid Handler, Andrew+, for Sample Preparation for LC-MS/MS-Based Quantitation of Immunosuppressants in Whole Blood
가톨릭대학교 의과대학 서울성모병원 진단검사의학과1, 녹십자의료재단 진단검사의학부2, 울산대학교 의과대학 서울아산병원 진단검사의학과3, 서울대학교 의과대학 분당서울대학교병원 진단검사의학과4, 연세대학교 의과대학 세브란스병원 진단검사의학과5, 순천향대학교 의과대학 순천향대학교부천병원 진단검사의학과6, 성균관대학교 의과대학 삼성서울병원 진단검사의학과7, 국립암센터 진단검사의학과8, 서울대학교 의과대학 서울대학교병원 진단검사의학과9
Department of Laboratory Medicine1, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul; Department of Laboratory Medicine2, Green Cross Laboratories, Yongin, Korea; Department of Laboratory Medicine3, University of Ulsan College of Medicine and Asan Medical Center, Seoul; Department of Laboratory Medicine4, Seoul National University Bundang Hospital and College of Medicine, Seongnam, Korea; Department of Laboratory Medicine5, Yonsei University College of Medicine, Seoul; Department of Laboratory Medicine and Genetics6, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon; Department of Laboratory Medicine and Genetics7, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; Department of Laboratory Medicine8, National Cancer Center, Seoul; Department of Laboratory Medicine9, Seoul National University Hospital, Seoul, Korea
Correspondence to:This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2023; 13(3): 199-204
Published July 1, 2023 https://doi.org/10.47429/lmo.2023.13.3.199
Copyright © The Korean Society for Laboratory Medicine.
Abstract
방법: 본 연구에서 저자들은 Andrew+ liquid handler를 활용하여 단백질 침전의 원리를 적용하는 반자동 검체 전처리 방법을 개발하고 이를 질량분석법에 접목시킨 4종 면역억제제(tacrolimus, sirolimus, everolimus, 그리고 cyclosporine A)의 치료적 약물농도 감시 검사를 평가하였다. 즉, 반자동 검체 전처리 방법의 유용성을 평가하고 총 163개의 임상 검체를 이용하여 검사 수행능을 분석하였으며 이를 수작업 검체 전처리법과 비교 평가하였다.
결과: Andrew+ liquid handler를 이용한 48개 검체 전처리법의 총 소요시간은 오프라인 원심분리 시간을 포함 총 55분이 소요되었다. 그러나 수작업 전처리법보다 hands-on time은 유의하게 감소되었다. 반복 정밀도와 검사실 내 정밀도는 검사실의 허용 가능한 범위 이내의 결과를 보였으며, 수작업 전처리법과 비교한 결과 두 검사법의 동등성을 확인하였다.
결론: 결론적으로 Andrew+ liquid handler를 활용하여 단백질침전법에 기반을 둔 반자동 검체 전처리 방법을 적용하여 질량분석법을 이용한 4종 면역억제제의 치료적 약물 모니터링에 이를 적용 가능하였다. Andrew+를 이용한 검체 전처리 방법은 재현성이 우수하며, 검사 절차가 표준화되어 있으며, 검사자의 시간 활용도를 개선해 주었다. 물론 전자동화 과정을 위해 최적화를 위한 노력이 더 필요하겠으나, 본 연구는 자동 액체 분주기를 접목하여 질량분석법을 이용한 임상검사의 접목이 굉장한 잠재력이 있음을 보여주었다.
Methods: We implemented a semi-automated sample preparation method based on protein precipitation, using an Andrew+ liquid handler, integrated with an LC-MS/MS-based TDM assay for four ISDs (tacrolimus, sirolimus, everolimus, and cyclosporine A). The feasibility and analytical performance were analyzed and compared with those of the manual sample preparation method using 163 clinical samples.
Results: The total execution time of Andrew+-based sample preparation method was 55 minutes (including off-line centrifugation) for 48 samples. However, the hands-on time was significantly reduced than that of the manual sample preparation method. The average repeatability and within-laboratory precision of the assay were acceptable (CV ≤10%), and the results of manual and Andrew+-based sample preparation methods were equivalent.
Conclusions: The sample preparation method using the Andrew+ liquid handler was successfully integrated with LC-MS/MS-based TDM assays for ISDs. Sample preparation using Andrew+ was reproducible, standardized, and enabled excellent utilization of technical staff time. Although further optimization for full automation is still pursued, this study indicates the potential of integrating liquid handers with LC-MS/MS-based assays in the clinical setting.
Keywords
INTRODUCTION
Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) optimizes their efficacy and minimizes toxicity in patients with organ transplantation and other autoimmune disorders [1]. The rationale behind this recommendation includes a narrow therapeutic index, large inter- and intra-individual pharmacokinetic variability, and severe consequences of both ISD overexposure and underexposure. For this purpose, clinical laboratories providing TDM of ISDs primarily utilize immunoassays (such as enzyme-multiplied, affinity chrome-mediated, chemiluminescent microparticle, and electrochemiluminescence immunoassays) or liquid chromatography/tandem mass spectrometry (LC-MS/MS). The analytical performance of LC-MS/MS-based assays is superior to that of immunoassays in terms of analytical accuracy and specificity [2], and LC-MS/MS is considered a reference method [3]. However, the labor-intensive and time-consuming sample preparation is a major drawback of using LC-MS/MS methods for TDM of ISDs.
Meanwhile, liquid handlers have been successfully used for the automation of workflows in clinical laboratories, including LC-MS/MS automation [4]. While most advancements in LC-MS/MS automation have originated from the pharmaceutical industry, the need for automation in medical diagnostic application of LC-MS/MS is nonetheless compelling. Hence, this study aimed to apply and optimize a liquid handler platform, Andrew+ (Waters, Milford, MA, USA) to the sample preparation workflow (based on protein precipitation) for LC-MS/MS-based TDM of four ISDs (tacrolimus, sirolimus, everolimus, and cyclosporine A) and analyze the relevant analytical performances related to the sample preparation method.
MATERIALS AND METHODS
1. Patient samples
All patient samples used in this study were residual samples collected for routine LC-MS/MS-based ISD assays. Whole blood samples were collected in Vacuette vacuum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) containing EDTA as anticoagulant. This study was reviewed and approved by the Institutional Review Board of Seoul St. Mary’s Hospital (KC22DISE0794), and a waiver of informed consent was granted.
2. Labware, chemicals, and reagents
Non-filtered, color-coded Optifit Tips compatible with Andrew+ were obtained from Sartorius (Goettingen, Germany). Ninety-six-well sample collection plates with 2 mL square wells, corresponding adhesive seals, and 96-well sample collection plates with 350 µL round wells were obtained from Waters. A six-column reagent reservoir was obtained from Agilent (Santa Clara, CA, USA).
LC-MS-grade methanol and formic acid were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA). Ammonium acetate and zinc sulfate heptahydrate were obtained from Sigma-Aldrich. Deionized water was generated using a Millipore Milli-Q Gradient Water Purification System (Molsheim, France). The 6PLUS1 multilevel whole blood calibrator sets and MassCheck whole blood controls were obtained from Chromsystems (Munich, Germany). The protein precipitation solution was 0.1 M ZnSO4. The internal standard working solution contained 10 ng/mL ascomycin (Sigma-Aldrich, Burlington, MA, USA) and 10 µg/mL cyclosporin D (United States Biological, Salem, MA, USA) in acetonitrile.
3. The liquid handling platform (Andrew+ pipetting robot)
1) The instrument
The liquid handling platform, Andrew+ pipetting robot (Waters), consists of a tool arm that can hold and charge up to four pipettes and a robotic arm for pipette and plate handling. Andrew+ also uses labware holders called dominos, and up to two full rows of dominos can be installed. In addition, by adding Device+ (Waters), processes such as heating, shaking, vacuum application, or labware transportation can be incorporated. The Andrew+ pipetting robot is controlled using the OneLab+ software (Waters) [5].
For the ISD assay, two multichannel Andrew Alliance Picus Pipettes (120 and 1,200 µL, Waters), a single-channel Andrew Alliance Picus Pipette (120 µL, Waters), two tip insertion system dominos, two conical microtube dominos, a deep well microplate domino, a microplate domino, and a microplate shaker+ device (Waters) were utilized (Fig. 1). The tip insertion system dominos were used to hold disposable pipette tips; the conical microtube dominos (for up to 24 samples each) were used for calibrators, controls, and samples; the deep well microplate domino was used for the six-column reagent reservoir that contained the protein precipitation and internal standard solutions; the microplate domino was used for the injection plate; and the microplate shaker+ device was used for the sample collection plate (reaction plate). An Eppendorf Centrifuge 5810R (Eppendorf SE, Hamburg, Germany) was used for off-line centrifugation.
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Figure 1. Andrew+ pipetting robot system layout. 1) A tool arm that holds and charges up to four pipettes, 2) a robotic arm for pipette and plate handling, 3) two pipette tip insertion system dominos, 4) two conical microtube dominos, 5) a deep well microplate domino, 6) a microplate domino, and 7) a microplate shaker device.
2) Workflow
The ISD assay workflow using Andrew+ consisted of three basic steps: sample transfer, protein precipitation, and sample analysis. A few manual steps were required, including the initial experimental setup, off-line centrifugation, and loading the injection plate onto the autosampler. The workflow process is described in detail in Fig. 2. The workflow and related pipetting parameters were designed and translated into OneLab executable scripts using an intuitive interface provided by the OneLab+ software. For off-line centrifugation, the plate was sealed and briefly mixed on a microplate shaker+ device and then centrifuged at 13,400×
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Figure 2. Workflow of the sample preparation process using the Andrew+ pipetting robot system configured for liquid chromatography/tandem mass spectrometry immunosuppressant analysis.
4. Ultra-performance liquid chromatography (UPLC)-MS/MS
An ACQUITY UPLC I-Class System (Waters) equipped with a Binary Solvent Manager and flow-through needle Sample Manager was used for chromatographic separation. For chromatographic separation, 20 µL of extracted sample was injected onto an ACQUITY UPLC HSS C18 SB column (1×100 mm, 1.8 µm particle size, 55.0°C column temperature, Waters). The mobile phase consisted of solution A (0.1% formic acid and 2 mM ammonium acetate in deionized water) and solution B (0.1% formic acid and 2 mM ammonium acetate in methanol) with the following linear gradient: initial, 50% B; 0.2–0.6 minutes, 50% B–100% B; 0.6–1.2 minutes, 100% B–50% B. The total run time was 1.8 minutes, with 0.4 mL/min constant flow rate.
UPLC was coupled to a Xevo™ TQD IVD MS (Waters). The electrospray ionization source was operated in the positive mode with the following parameters: capillary voltage, 3 kV; cone voltage, 34 V; source temperature, 150°C; desolvation temperature, 400°C; nitrogen desolvation gas flow, 1000 L/h; and nitrogen cone gas flow, 10 L/h. The compounds were detected via multiple reaction monitoring mode employing the following ion transitions:
5. Method evaluation
The total process time, hands-on time, feasibility of integration with the LC-MS/MS-based TDM of ISDs, and analytical performance of Andrew+-based UPLC-MS/MS method were evaluated. Three levels of MassCheck whole blood-based quality controls (ChromSystems) were utilized for precision evaluation. In addition, 163 samples (45 tacrolimus, 37 sirolimus, 36 everolimus, and 44 cyclosporine A), encompassing the target range of respective ISDs (i.e., tacrolimus 5–20 ng/mL, sirolimus 5–15 ng/mL, everolimus 3–8 ng/mL, cyclosporine A 150–300 ng/mL), were analyzed using both the manual and Andrew+-based sample preparation methods, and the results were compared. Analyse-it for Microsoft Excel (Analyse-it Software, Ltd., Leeds, UK) was used for data processing. Passing–Bablok nonparametric linear regression was used for method comparison.
RESULTS
The total execution time of the sample preparation method developed using Andrew+ pipetting system for quantification of four ISDs was 55 minutes (including 20 minutes for off-line centrifugation) for 48 samples (Table 1). Since a significant amount of time was allotted for off-line centrifugation (10 and 20 minutes for the manual and Andrew+-based preparation methods, respectively), it is safe to say that the total time required for preparing 48 samples was comparable between the manual and Andrew+ preparation methods. Meanwhile, the hands-on time was significantly reduced from 27 to 2 minutes when using the Andrew+ pipetting system.
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Table 1 Comparison of total and hands-on times using manual vs. automated sample preparation methods used in this study for 48 samples
Protocol steps Manual Automated Sample transfer Calibrator (N = 6), QC (N = 3), blank (N = 2), and sample (N = 39) transfer 6 min 27 min Protein precipitation 0.1 M ZnSO4 addition (and mixing) 8 min 2 min Internal standard solution addition (and mixing) 10 min 3 min Centrifugation 10 min 20 min Sample Transfer Supernatant transfer 3 min 3 min Total time 37 min 55 min Total hands-on time 27 min 2 min
The repeatability and within-laboratory precision of the assay assessed at three separate QC levels are shown in Table 2. Repeatability and within-laboratory precision were both acceptable and fulfilled the recommended performance criteria for imprecision, where applicable (CV ≤10% and ≤5% for 50 and 300 ng/mL cyclosporine A, respectively) [6].
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Table 2 Analytical imprecision of the Andrew+-based liquid chromatography/tandem mass spectrometry assay
Analyte QC Level Mean (ng/mL) Repeatability* Within-laboratory Precision† SD (ng/mL) CV% SD (ng/mL) CV% Tacrolimus Level 1 2.6 0.18 7.1 0.24 9.2 Level 2 7.0 0.31 4.4 0.41 5.8 Level 3 14.8 0.51 3.5 0.59 4.0 Sirolimus Level 1 2.8 0.26 9.6 0.27 10.0 Level 2 10.1 0.66 6.5 0.93 9.2 Level 3 19.3 1.06 5.5 1.40 7.2 Everolimus Level 1 2.5 0.22 8.8 0.28 11.1 Level 2 4.7 0.39 8.3 0.43 9.2 Level 3 9.0 0.41 4.6 0.55 6.1 Cyclosporine A Level 1 46.9 1.48 3.1 2.45 5.2 Level 2 248.3 4.24 1.7 5.37 2.2 Level 3 492.5 8.91 1.8 11.48 2.3 *Repeatability and †within-laboratory precision were assessed using three QC materials assayed over 5 days, with two runs per day, and five replicates per run: a 5×2×5 design.
The equations of the Passing–Bablok regression lines for all four ISDs are shown in Table 3. The estimates of parameters (slope and intercept with their lower and upper limits of the 95% confidence intervals) using the Passing–Bablok regression between the manual and Andrew+ sample preparation methods are shown in Table 2. For all four ISDs, the 95% confidence intervals of the slope and intercept encompassed 1 and 0, respectively; hence, the statistical differences between the results of the manual and Andrew+-based sample preparation methods integrated with LC-MS/MS were considered insignificant. Moreover, the bias and 95% CI of bias estimate, calculated from the regression line at relevant medical decision points of each ISDs (i.e., tacrolimus 5 ng/mL, sirolimus 5 ng/mL, everolimus 3 ng/mL, cyclosporine A 150 ng/mL) were within the prespecified acceptance criteria of 10%.
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Table 3 The estimates of Passing–Bablok regression between the manual (x) and Andrew+-based sample preparation (y) methods
Analyte Estimate 95% CI Tacrolimus (n = 45) Intercept -0.6621 -1.493 to 0.2000 Slope 1.103 1.000 to 1.213 Spearman’s ρ* 0.953 0.913 to 0.974 Sirolimus (n = 37) Intercept 0.2000 -0.8571 to 1.183 Slope 1.0000 0.8280 to 1.149 Spearman’s ρ 0.952 0.907 to 0.976 Everolimus (n = 36) Intercept 0.1778 0 to 0.5392 Slope 0.9751 0.8824 to 1.032 Spearman’s ρ 0.975 0.950 to 0.988 Cyclosporine A (n = 44) Intercept -3.9830 -14.07 to 4.396 Slope 0.9846 0.8929 to 1.129 Spearman’s ρ 0.985 0.972 to 0.992 *Spearman’s rank correlation coefficient.
DISCUSSION
LC-MS/MS method is increasingly used for TDM of ISDs in clinical laboratories, mainly because of its high analytical specificity and multiplexing capability [7]. Published sample preparation methods for LC-MS/MS-based ISD assays include protein precipitation, liquid–liquid extraction [8], and online solid-phase extraction [9]. However, in many laboratories, the samples for LC-MS/MS assays for ISDs are prepared manually [10]. The Andrew+ pipetting robot, a versatile liquid handling system for increasing automation in the sample preparation steps for LC-MS/MS assays, has been recently introduced. While an application note from Andrew+ for semi-automated sample preparation for LC-MS/MS-based ISDs is available [11], to the best of our knowledge, this is the first report of using the Andrew+ pipetting system for LC-MS/MS-based TDM assays for ISDs using clinical samples. In this report, substituting manual extraction by the semi-automated protein precipitation method using the Andrew+ pipetting system allowed reproducible and standardized sample preparation workflow with less hands-on time for whole blood samples and provided comparable results for LC-MS/MS-based ISD quantification.
One of the challenges in adopting a liquid handler for sample preparation for TDM of ISDs is maintaining homogeneity of the whole blood sample, since the ISDs are partitioned into erythrocytes and the cellular components sediment within minutes when the sample is at rest. To ensure whole blood homogeneity, we applied resuspension procedures using repeated aspiration/dispensing, and the results were comparable to those of the manual preparation method, which utilizes mixing by agitation of primary blood collection tubes. In addition, we modified the off-line centrifugation force of the OneLab protocol of 2,000×
Equivalence in analytical performance (such as precision and comparison) was observed using Andrew+ for transferring samples, calibrators, QCs, and reagents, as well as adding internal standard and protein precipitation solutions and transferring extracted supernatants. However, there is still room for improvement, and further optimization could be pursued to achieve the full potential of automated sample preparation using the Andrew+ platform. For example, off-line centrifugation is a major non-automated step in the present workflow, necessitating a significant amount of time (20 minutes) and additional hands-on time. Alternatives to centrifugation after protein precipitation that can increase the degree of automation include vacuum filtration, which is a viable solution for full automation, particularly when a new vacuum pump, extraction+device, controlled via the OneLab Software, have been introduced. Another feature that could be optimized is the option to use primary blood collection tubes, with perhaps a primary tube cap piercing feature that could replace manual de-capping and sample transfer.
In summary, the results demonstrate the feasibility of integrating the Andrew+ liquid handler with LC-MS/MS-based TDM assays for ISDs. Sample preparation based on the protein precipitation method using Andrew+ is reproducible, provides standardized workflow, and significantly reduces hands-on time, which is one of the key drivers of adoption of automated sample preparation methods for LC-MS/MS. Moreover, the assay performance was equivalent to that of the manual protein extraction method. Although further optimization is being pursued, this study indicates the potential of integrating relatively simple liquid handlers with LC-MS/MS-based assays in the clinical laboratory.
Conflicts of Interest
Acknowledgments
References
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