Analysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry

doi:10.1016/j.jchromb.2009.03.024

Journal of Chromatography B

Volume 877, Issues 14–15, 15 May 2009, Pages 1595-1598

https://doi.org/10.1016/j.jchromb.2009.03.024 Get rights and content

Abstract

Dried blood spot sampling is a promising and patient friendly alternative for venous sampling. A liquid chromatography tandem mass spectrometer assay was developed for analyzing cyclosporin A in dried blood spots. Linearity ranged from 25 to 1440 μg/L. Within and between run accuracy and precision were within limits. The developed assay has a negligible matrix-effect and a recovery of 97%. The dried blood spots were stable during a period of at least 17 days in the refrigerator. The developed assay is suitable for analyzing cyclosporin A in dried blood spots.

Introduction

Cyclosporin A (CsA) (Fig. 1) is an immunosuppressant drug which specifically and reversibly inhibits the proliferation of T-lymphocytes. CsA is indicated for prohibiting rejection of transplanted organs, graft versus host disease and severe psoriasis. Because of its major side-effects such as nefrotoxicity, disruption of liver function and convulsions, and its variance in inter- and intra-individual bio-availability Therapeutic Drug Monitoring (TDM) of CsA is necessary [1]. Dosage of CsA is due to the different bio-availability. Therapeutic drug ranges vary per transplant clinic and type of transplantation. The minimum therapeutic drug range in bone marrow transplantation is 200–350 μg/L.

As published earlier for tacrolimus, dried blood spot (DBS) sampling is a promising and patient friendly alternative for venous sampling [2], [3], [4]. DBS samples are made with capillary blood, obtained from a fingerprick with an automatic lancet. Blood drops are applied to the sampling paper which is, after drying at ambient temperature, mailed by postal service to our laboratory. At our laboratory a disk is punched out from the blood spot for further sample preparation and analysis.

A transition from immunoassay to liquid chromatography tandem mass spectrometry (LC–MS/MS) was recently made for analyzing CsA in venous blood [5]. LC–MS/MS is more specific and sensitive compared to immunoassays and does not require the use of radio-activity in comparison to radioimmunoassays [6]. Usage of the DBS sampling method for CsA has been described earlier for radioimmunoassay and monoclonal antibody techniques [7], [8], however no method for analyzing DBS samples with LC–MS/MS was described earlier. We developed a simple, precise and accurate method for analyzing CsA DBS samples with LC–MS/MS.

Section snippets

Chemicals

Two different batches cyclosporin A (Biochemika ≥98.5%) were purchased from Sigma–Aldrich (Zwijndrecht, The Netherlands). Internal standard cyclosporin D (CsD) (>99%) was purchased from Eton Bioscience Inc. (San-Diego, USA). Methanol (HPLC-grade) was purchased from Biosolve (Valkenswaard, the Netherlands). Formic acid (pro analysi), zincsulfate (Titrisol) and ammonium acetate (pro analysi) were purchased from Meck BV (Darmstadt, Germany). Sampling paper (903 Protein Saver Card) was purchased

Matrix-effect and recovery

As matrix-effects in LC–MS/MS analysis can be of major influence [9], [10] sample preparation should be as clean as possible. Therefore acetonitrile and methanol were tested as extraction solution, these tests showed recoveries of 20–50% (data not shown). Although there is no clear minimal recovery described [9], [10], [11] it is suggested to be at least 70%. In literature a sample preparation procedure with recoveries of ≥90% was found [8]. In the described sample preparation procedure blood

Discussion and conclusion

As shown in Table 1 all validated parameters are within limits. The developed sample pretreatment procedure has a small, negligible matrix-effect and high extraction recovery which is, more importantly, consistent. The within and between run accuracy is sufficient for LLOQ, low, medium and high quality controls. The within and between run precision was well within limits for all quality controls.

We did not detect an influence of HT on CsA concentrations in the range of 0.30 HT to 0.59 HT.

The

Acknowledgment

We would like to thank Dr. P.M. Edelbroek from the Epilepsy Institute SEIN for his support and advice.

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Cited by (68)

Application of non-contact hematocrit prediction technologies to overcome hematocrit effects on immunosuppressant quantification from dried blood spots
2023, Talanta
Fully automated dried blood spot (DBS) analysis for therapeutic drug monitoring (TDM) of the immunosuppressants tacrolimus, sirolimus, everolimus and cyclosporin A suffers from a so-called hematocrit (hct) effect. This effect is related to the analysis of a partial DBS punch and extractability differences imposed by blood with different hcts. As this is intrinsic to automated DBS analysis, this poses a serious drawback for accurate immunosuppressant quantification. Knowledge of a sample's hct allows to correct the derived immunosuppressant concentrations for this effect. Unfortunately, when using the DBS approach for sampling at patients' homes, this hct will typically not be available. The aim of this study was to investigate the validity of a correction algorithm during fully automated DBS analysis of immunosuppressants, based on knowledge of the DBS′ hct, obtained via two distinct non-contact hematocrit prediction strategies, using either near-infrared (NIR) or ultra-violet/visible (UV/VIS) spectroscopy.
For tacrolimus, sirolimus, everolimus, and cyclosporin A, 48, 47, 58 and 48 paired venous whole blood and venous DBS patient samples were collected, respectively, and analyzed using an automated DBS-MS 500 HCT extraction unit coupled to a liquid chromatography tandem mass spectrometry system. Additionally, for all 201 samples the hct of the DBS was predicted based on NIR and UV/VIS spectroscopy. For tacrolimus and cyclosporin A, both hct prediction strategies allowed for adequate correction of the hct effect. Also for sirolimus and everolimus the results greatly improved after hct correction, although a hct bias remained for sirolimus and for everolimus a slightly significant hct effect was observed after NIR- and UV/VIS-based correction. Application of both hct prediction strategies ensured that clinical acceptance limits (i.e. ≥ 80% of the samples within 20% difference compared to whole blood) were met for all analytes.
In conclusion, we demonstrated that non-contact hct prediction strategies, applied in tandem with fully automated DBS analysis, can be used to adequately correct immunosuppressant concentrations, yielding a good agreement with whole blood.
Dried blood microsampling-assisted therapeutic drug monitoring of immunosuppressants: An overview
2023, Journal of Chromatography A
Citation Excerpt :
For most TDM applications, including immunosuppressant drug monitoring, cellulose type filter papers are used, either untreated or treated. For immunosuppressants monitoring, Whatman 903 [9,30,32,39,41,42,46,48,50–52,54–63,68,74] is most commonly used, followed by Whatman FTA DMPK-C [20,64–66], Whatman 10535097 [8,45,51,67,103] and Perkin Elmer 226 filter paper [40,69,70]. The influence of the type of filter paper was illustrated in a study by Koster et al., who performed an in-depth investigation of the effect of different filter papers on the quantification of immunosuppressants [71].
In the field of solid organ transplantation, chemotherapy and autoimmune disorders, treatment with immunosuppressant drugs requires intensive follow-up of the blood concentrations via therapeutic drug monitoring (TDM) because of their narrow therapeutic window and high intra- and inter-subject variability. This requires frequent hospital visits and venepunctures to allow the determination of these analytes, putting a high burden on the patients. In the context of patient-centric thinking, it is becoming increasingly established that at least part of these conventional blood draws could be replaced by microsampling, allowing home-sampling and increasing the quality of life for these patients. In this review we discuss the published methods - mostly using liquid chromatography coupled to tandem mass spectrometry - that have utilized (volumetric) dried blood samples as an alternative for conventional liquid whole blood for the TDM of immunosuppressant drugs. Furthermore, some pre-analytical considerations using DBS or volumetric alternatives are considered, as well as the applicability on clinical samples. The implementation status in clinical practice is also discussed, including (1) the cost-effectiveness of this approach compared to venepuncture, (2) the availability of multiplexed methods, (3) the status of harmonization and (4) patient perception. A brief perspective on potential future developments for the dried blood-based TDM of immunosuppressant drugs is provided, by considering how obstacles for the implementation of these strategies into clinical practice might be overcome.
Quantification of immunosuppressants from one 3.2 mm dried blood spot by a novel cold-induced phase separation based LC-MS/MS method
2022, Analytica Chimica Acta
Dried blood spots (DBS) have been regarded as a promising alternative for therapeutic drug monitoring (TDM) of immunosuppressants (ISDs) for over fifteen years. Nonetheless, there are still three main issues impeding its preference: (i) the requirement of relatively large disc; (ii) the controversial and intricate desorption approaches; (iii) the lack of feasible extraction strategies. For improvement, this work described a new LC-MS/MS method realizing quantification of four ISDs from one piece of 3.2 mm DBS. During sample pretreatment, a modified approach (infiltrating the DBS in pure water before adding acetonitrile and zinc sulfate as protein-precipitators) was developed to completely dissociate the targets from filter paper. Afterward, effective enrichment and purification of the targets were achieved by using cold-induced phase separation technique. Benefiting from these novelties, the method exhibited satisfying throughput (15 min for sample preparation), applicability (consuming only one 3.2 mm disc), reliability (82.3–107.8% for accuracy and <14.3% for precision) and sensitivity (lower limit of quantification of 0.5, 7.6, 0.7 and 0.8 ng mL⁻¹ for tacrolimus, cyclosporine A, everolimus and sirolimus, respectively). Without hematocrit correction, the method showed favorable interchangeability to the certified whole blood method through analyzing 120 paired clinical samples. By taking ±20% of the mean as the limit of acceptance for cross validation, over 90% of the detection met the criterion. It can be expected the developed method is able to further promote the popularization of DBS-based TDM for ISDs in practice.
Quantitative dried blood spot analysis for metallodrugs by laser ablation-inductively coupled plasma-mass spectrometry
2019, Journal of Trace Elements in Medicine and Biology
A quantitative dried blood spot (DBS) method based on direct sampling by means of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is presented. Gadolinium-based contrast agents were used as model metallodrugs with a significant relevance for pharmaceutical applications. Challenges regarding the ablation of the complex blood-filter matrix were characterized and successfully addressed by a thorough adaption of the laser ablation conditions. Especially the laser fluence was optimized with respect to the particle size distribution of the generated aerosol as monitored by an optical particle counter. Thus, generation of micrometer-sized particles could be minimized in favor of smaller particles increasing the transport efficiency of the DBS ablation aerosol to the plasma and the recorded signal stability. Inhomogeneous blood drying on the porous filter paper could be compensated by the addition of an internal standard prior to blood spotting. To preserve the advantages of DBS sampling, such as small blood volumes and minimal invasiveness, the combined use of DBS and a capillary blood sampling system is demonstrated. By placing the internal standard into the capillary prior to blood sampling, a simple workflow usable for clinical application was implemented. The applicability of the developed method, achieving limits of detection and quantification in the low μg L⁻¹ range and covering a linear range of over four orders of magnitude, was demonstrated for blood samples containing different concentrations of the gadolinium contrast agents gadopentetate and gadoterate.
LC–MS/MS method for the simultaneous analysis of seven antimalarials and two active metabolites in dried blood spots for applications in field trials: Analytical and clinical validation
2018, Journal of Pharmaceutical and Biomedical Analysis
In epidemiological studies, antimalarials measurements in blood represent the best available marker of drugs exposure at population level, an important driver for the emergence of drug resistance. We have developed a liquid chromatography-tandem mass spectrometry method (LC–MS/MS) for the simultaneous quantification of 7 frequently used antimalarials (amodiaquine, chloroquine, quinine, sulfadoxine, pyrimethamine, mefloquine, lumefantrine) and 2 active metabolites (N-desethyl-amodiaquine, desbutyl-lumefantrine) in 10-μl dried blood spots (DBS). This sampling approach is suitable for field studies wherein blood samples processing, transportation and storage are problematic. Sample preparation included extraction from a 3 mm-disk punched out of the DBS with 100-μl of methanol + 1% formic acid containing deuterated internal standards for all drugs. Good performances were achieved in terms of trueness (−12.1 to +11.1%), precision (1.4–15.0%) and sensitivity, with lower limits of quantification comprised between 2 ng/ml (sulfadoxine) and 20 ng/ml (chloroquine, quinine, pyrimethamine, mefloquine, lumefantrine and desbutyl-lumefantrine). All analytes were stable in DBS kept for 24 h at room temperature and at 37 °C. The developed assay was applied within the frame of a pharmacokinetic study including 16 healthy volunteers who received a single dose of artemether-lumefantrine. Lumefantrine concentrations in plasma and in DBS were highly correlated (R = 0.97) at all time points, confirming the assumption that lumefantrine concentrations determined in DBS confidently reflect blood concentrations. The blood/plasma ratio of 0.56 obtained using the Bland-Altman approach (and corresponding to the slope of the linear regression) is in line with very low penetration of lumefantrine into red blood cells. This sensitive multiplex LC–MS/MS assay enabling the simultaneous analysis of antimalarials in DBS is suitable for epidemiological studies in field conditions.
Clinical feasibility of dried blood spots: Analytics, validation, and applications
2016, Journal of Pharmaceutical and Biomedical Analysis
Dried blood spots (DBS) sampling and their specific advantages are becoming common in analytical and clinical routine. Being first established for metabolic disorder screening in neonates, its use emerged to a broad spectrum of clinical applications. Although DBS are easily generated, the conduction of specific analytical and clinical validation procedures should be obligatory when implementing DBS for clinical purposes, e.g. therapeutic drug monitoring or clinical drug trials. A respective recommendation has already been published by the European Bioanalysis Forum. Since no official guidelines are present, investigators are currently free in DBS procedure development and validation. This review summarizes and discusses published clinical validation procedures in relation to their applications to highlight the clinical feasibility of DBS.

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Short communicationAnalysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry

Abstract

Introduction

Section snippets

Chemicals

Matrix-effect and recovery

Discussion and conclusion

Acknowledgment

Clin. Ther.

J. Immunol. Methods

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

Clin. Chim. Acta

Br. J. Clin. Pharmacol.

Transplantation

Short communication
Analysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry