MSACL 2017 US Abstract

Capillary Blood Collected on Volumetric Absorptive Micro Sampler for Therapeutic Drug Monitoring of Hydroxychloroquine

Ying Qu (Presenter)
Exagen Diagnostic, Inc.

Bio: Dr. Ying Qu is a senior scientist in Exagen Diagnostics Inc. She earned her B.S. and M.S. in Chemistry specialized in Analytical Chemistry from Lanzhou University in China; Ph.D. in Neuroscience from University of Leuven in Belgium; postdoctoral training in Neuropharmacology from National Institutes of Health in USA. Her research work is focused on applying analytical technology to solve the challenge problem in life science which resulted over 30 publications in both field of analytical chemistry and life science. She has used LC-MS/MS as main technology to support several drug discovery and development programs in Johnson and Johnson, Amylin Pharmaceuticals Inc. She currently leads the diagnostic project of clinical mass spectrometer.

Authorship: Ying Qu(1), Kelley Brady(1), Robert Apilado(1), Puja Chitkara(2), Smitha Reddy(3), Claudia Ibarra(1), Roberta Vezza Alexander(1), Thierry Dervieux(1)
(1)Research and Development, Exagen Diagnostics Inc., Vista, CA, USA; (2). Arthritis Care and Research Center, Poway, CA, USA; (3). Center for Arthritis and Rheumatologic Excellence, Chula Vista, CA, USA

Short Abstract

Volumetric absorptive microsampling (VAMS) blood collection method was successfully applied for the first time to the therapeutic drug monitoring of hydroxychloroquine (HCQ) in rheumatoid arthritis patients. LC-MS/MS workflow for analysis of VAMS sample was developed and validated. Drug concentrations of HCQ and its metabolites in capillary blood on VAMS were compared to those in venous blood. Our results established that VAMS is a simple and accurate sampling device that delivers the benefits of dried blood spots (DBS) sampling while overcoming the issues associated with hematocrit and homogeneity.

Long Abstract

INTRODUCTION

Therapeutic drug monitoring (TDM) is usually performed using 5-10 ml of venous blood collected by venipuncture in anticoagulant-containing vacutainer tubes. Refrigeration during storage and shipment requires logistics management, and translates into significant costs. In addition, it may be difficult for vulnerable populations, such as young children and the elderly, to provide the large amount of venous blood required. As an alternative sample collection method, dried blood spot (DBS) cards have been used in pediatric populations since 1963 (1). DBS cards require a few droplets of capillary blood (20-50 µl) and, when dry, can be shipped at room temperature, thereby simplifying shipment logistics. However, blood does not spread homogeneously on the DBS card material, and the size of the spot is influenced by the blood viscosity which, in turn, is dependent on the hematocrit (HCT) (2, 3). These limitations may lead to inaccurate results and, thus, render DBS cards non amenable to TDM. A novel blood sampling device, termed volumetric absorptive micro-sampler (VAMS), has been designed to deliver the benefits of DBS cards while overcoming the issues associated with HCT and homogeneity. The VAMS consists of an absorbent polymeric tip that, when dipped into blood, absorbs a fixed volume (nominally 10 µl) by capillary action. Experiments with blood of different HCT (20-65%) demonstrated that tips absorb 10.6 ± 0.4 µl independently of the HCT (4, 5). The VAMS device is on file with the FDA as a class 1 device.

Hydroxychloroquine (HCQ) is widely used for the treatment of patients with autoimmune rheumatic diseases (6, 7). Because nonadherence is common, TDM of HCQ is particularly important to identify poor or not adherent patients, and to optimize treatment in patients at risk of disease exacerbation (8-11). As VAMS could simplify TDM of HCQ, we investigated the use of VAMS for this application. We conducted a study in patients with rheumatoid arthritis (RA) treated with HCQ; capillary blood was collected on VAMS by finger prick, and venous blood was drawn in EDTA-containing tubes. We developed and validated a LC-MS/MS method to measure the concentration of HCQ and its metabolites [desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ)] in capillary blood on VAMS and venous blood samples.

METHODS

From November 2015 to August 2016, we enrolled 54 patients with rheumatoid arthritis and receiving HCQ therapy at the dosage of 200-400 mg/day. Capillary blood was collected by finger prick, and venous blood was drawn in EDTA-containing tubes. In a subset of patients (n=44), capillary blood was collected not only on VAMS, but also on DBS cards. Capillary blood samples were dried at room temperature, shipped to the analytical site, and extracted using vortexation followed by protein precipitation. VAMS extracts were separated on a Kinetex C8 column and detected by TSQ-Quantiva triple quadrupole mass spectrometer with a heated electrospray ionization source in the positive mode.

RESULTS

LC-MS/MS Method Development and Validation. The calibration range of the LC-MS/MS method was 10-2000 ng/mL for HCQ and DHCQ, and 4-400 ng/mL for DCQ and BDCQ. The intra-day and inter-day precision ranged from 1.8 to 10.0%. The lower limit of quantification (LLOQ) was 10 ng/mL for HCQ and DHCQ, and 4 ng/mL for DCQ and BDCQ. No interference from endogenous or exogenous substances was observed in all chromatograms of VAMS extracts. There was no carry-over and no matrix effect for HCQ and its three metabolites.

Method Comparison. Concentrations of HCQ and its metabolites were measured in parallel by HPLC-FLD (11) and LC-MS/MS. Deming regression analysis yielded slope of 0.96 for HCQ; correlation coefficient was 0.98. For DHCQ and DCQ, correlation coefficients were 0.98 and 0.99, and slopes were 0.98 and 1.07, respectively. These results indicate an excellent correlation between the two methods.

VAMS Sampling of Patients. Capillary blood from 44 patients was collected on both VAMS and DBS cards. The minimal volume of one drop of blood to fill one spot of a DBS card is approximately 20–50 µL. About 20% of the patients failed to produce enough blood to cover a 6 mm-diameter spot on the DBS card, as required by the sensitivity of LC-MS/MS method. However, VAMS collection was successful for all 44 patients (100%), possibly because of the smaller blood volume required (10 µL).

Comparison Between Capillary Blood on VAMS and Venous Blood. Concentrations of HCQ and its metabolites were measured in parallel in capillary blood on VAMS and in venous samples of 54 HCQ-treated patients. Comparison of the concentration of HCQ in capillary blood on VAMS and venous blood yielded a correlation coefficient of 0.97 and Deming slope of 1.08. Comparison of DHCQ, DCQ, and BDCQ yielded correlation coefficients of 0.97, 0.95, and 0.96 and Deming slopes of 1.04, 1.11, and 1.07, respectively. These results demonstrate that capillary blood collected on VAMS is a valuable alternative to venous blood for TDM of HCQ and its metabolites.

Stability of Dried Capillary blood in VAMS. Ambient temperature stability of HCQ in dried capillary blood on VAMS was evaluated using samples from 32 HCQ-treated patients. Two VAMS were collected from each patient, dried at room temperature, and extracted 3 and 10 days after collection, respectively. LC-MS/MS analysis was performed immediately after sample extraction. Regression analysis of HCQ, DHC, DCQ, and BDCQ yielded correlation coefficients of 0.96, 0.95, 0.90, and 0.93, respectively, and Deming slopes of 1.01, 1.09, 1.09, and 1.13, respectively. These results indicate that HCQ, DHCQ, DCQ, and BDCQ are stable in VAMS at ambient temperature for up to 10 days.

High ambient temperature stability of HCQ in dried blood on VAMS was evaluated using samples from 27 patients, after preparing VAMS from venous blood. VAMS were kept at 22°C and 50°C (122° F, which corresponds to extreme hot weather in the US) for 24 hours, respectively, before extraction. Regression analysis of HCQ, DHCQ, DCQ, and BDCQ yielded correlation coefficients of 0.98, 0.97, 0.99, and 0.97, respectively, and Deming slopes of 0.92, 0.97, 0.92, and 0.91, respectively. These results indicate that HCQ, DHCQ, DCQ and BDCQ in dried blood on VAMS are stable at high ambient temperature (122° F) for 24 hours, implying that samples can be processed after overnight transport in extreme hot weather.

In this population of RA patients, capillary blood levels of HCQ were 990 ± 92 ng/ml (average ± SEM), DHCQ levels were 585 ± 53 ng/ml, DCQ levels were 77 ± 7 ng/ml, and BDCQ levels were 42 ± 4 ng/ml. 18.5% of patients presented with HCQ levels below 200 ng/ml, thereby indicating noncompliance to treatment.

CONCLUSION

To our knowledge, this is the first study demonstrating that VAMS can be used to collect capillary blood for TDM. Concentrations of HCQ and its metabolites in capillary blood collected on VAMS are comparable to those measured in venous blood. Therefore, implementation of VAMS in an analytical workflow may simplify sample collection, transport, storage, and processing, adding flexibility to data collection for TDM. The small amount of blood required for VAMS may decrease patient burden, especially in pediatric or elderly populations. Furthermore, VAMS enable sample collection in the patient’s home setting or in resource-limited areas where sample collection by venipuncture may be challenging.


References & Acknowledgements:

Acknowledgements: We acknowledge the patients for their participation in the study and the medical staff and coordinators at each sites. We also thank Neoteryx for providing VAMS.

REFERENCE

1. R. Guthrie, A. Susi, A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics 32, 338-343 (1963).

2. P. Denniff, N. Spooner, The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis 2, 1385-1395 (2010).

3. M. Wagner, D. Tonoli, E. Varesio, G. Hopfgartner, The use of mass spectrometry to analyze dried blood spots. Mass Spectrom Rev 35, 361-438 (2016).

4. N. Spooner et al., A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit. Bioanalysis 7, 653-659 (2015).

5. P. Denniff, N. Spooner, Volumetric absorptive microsampling: a dried sample collection technique for quantitative bioanalysis. Analytical chemistry 86, 8489-8495 (2014).

6. M. Somer et al., Influence of hydroxychloroquine on the bioavailability of oral metoprolol. Br J Clin Pharmacol 49, 549-554 (2000).

7. T. Munster et al., Hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis. Arthritis Rheum 46, 1460-1469 (2002).

8. N. Costedoat-Chalumeau et al., Hydroxychloroquine in systemic lupus erythematosus: results of a French multicentre controlled trial (PLUS Study). Ann Rheum Dis 72, 1786-1792 (2013).

9. N. Costedoat-Chalumeau et al., Low blood concentration of hydroxychloroquine is a marker for and predictor of disease exacerbations in patients with systemic lupus erythematosus. Arthritis Rheum 54, 3284-3290 (2006).

10. N. Costedoat-Chalumeau et al., Very low blood hydroxychloroquine concentration as an objective marker of poor adherence to treatment of systemic lupus erythematosus. Ann Rheum Dis 66, 821-824 (2007).

11. Y. Qu et al., Development and validation of a clinical HPLC method for the quantification of hydroxychloroquine and its metabolites in whole blood. Future Science OA 1, DOI 10.4155/fso.4115.4124 (2015).


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesExagen Diagonistic Inc.
Board Memberno
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes