Work

Drug analysis: Comparison between dried plasma spots and liquid plasma samples of trauma patients from Cameroon—A feasibility study

Year: 2023

Type: article

Abstract: Analysis of drugs in blood samples in some low- and middle-income countries (LMIC) can be difficult or practically impossible. Laboratories in those countries might not have the financial possibilities to buy expensive equipment, such as immunological test equipment for drug screening, equipment for high-performance liquid chromatography or gas chromatography for more specific drug analysis, or mass spectrometric detectors for specific identification. Also, the maintenance and repair of these types of equipment might be difficult because of a lack of local spare parts or maintenance personnel. The import of reagents and calibration standards might be difficult if the manufacturers do not have representatives in the country. Also, some countries do not allow the import of calibration standards for illicit drugs, so quantitation may be impossible. Due to the challenges with the analysis of drugs in blood samples, many countries rely on the analysis of urine samples instead of blood, often by using simple test strips (dipsticks) or point-of-care cups to determine the presence of drugs.1, 2 Tests of that type may not have good diagnostic accuracy as cross-reactions with other substances may occur.2, 3 Positive test results in urine only tell us that the drug has most likely been used. Therefore, blood, serum or plasma samples are preferred for therapeutic drug monitoring and for the analysis of drugs when assessing possible impairment or toxicological effects. It may then be necessary to ship samples to laboratories abroad for drug testing. There are strict regulations that need to be followed when shipping blood samples because they may be infectious. This includes the presence of absorbent material in the shipping container; an appropriate coolant; a water-protective inner package; accurate labelling; an import permit, and sometimes an export permit; and documentation.4, 5 Failure to comply with the appropriate shipping guidelines may result in delays or failures in delivery. A good alternative is to prepare dried matrix spots of whole blood, serum, or plasma that can be sent by regular mail or courier services. Dried matrix spots are much less contagious and therefore safer, and they can be stored at room temperature for several days without any significant degradation.6-8 The packaging and shipment of dried matrix spots are far simpler than with liquids, consisting of a water-protective package as the inner, primary container that holds the dried sample; next, a secondary, watertight envelope for additional protection; and finally, a sturdy, high-quality outer paper package.9 Dried blood or plasma spots have been used in epidemiological studies in LMICs, for example, in studies of infectious diseases6, 10, 11 and in therapeutic drug monitoring.12, 13 Dried samples are also suitable for analysis of drugs of abuse.7, 14, 15 Several devices are available to collect and ship dried matrix spots; some of them are designed to collect defined sample volumes. These devices might be too expensive for use in resource-limited settings. An inexpensive alternative could be to use large filter paper sheets for multiple samples. The use of homemade paper samples has previously been explored for protein analysis.16 In a research project on patients injured in road traffic crashes in Cameroon, it was decided to ship frozen liquid plasma samples (LPSs) abroad for qualitative drug testing. The first sample shipment was stranded at an airport and never arrived at the laboratory. It was therefore decided to ship dried plasma spots (DPSs) abroad for testing. Several months later, the project team found an alternative shipping carrier for frozen plasma samples, which were successfully delivered to the analytical laboratory. Here, we describe a simple procedure for the preparation and shipment of DPS and the results of drug testing compared with drug findings in frozen LPSs. Trauma patients admitted to Hôpital Laquintinie de Douala (Douala, Cameroon) after involvement in road traffic crashes were asked to participate in an anonymous and voluntary study that included testing for alcohol in breath and drugs in plasma. Participating patients gave informed consent. The study was approved by the National Ethical Committee for Research for Human Health, Cameroon, with Approval No. 2018/07/1080/CE/CNERSH/SP. Blood samples were collected in 4 ml vacutainer tubes containing 2.0 mg/ml potassium oxalate and 2.0 mg/ml sodium fluoride (Becton, Dickinson and Company, Franklin Lakes, NJ, United States). The samples were centrifuged at 2,000 rpm for 10 min at 4°C, and aliquots of plasma samples were frozen at −80°C at the National Public Health Laboratory (Yaoundé, Cameroon). After thawing, 4 × 15 μl plasma was applied onto a Whatman CF12 filter paper sheet (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom) cut into a size of 21 × 28 cm. A pattern to help with the application of samples was printed on each sheet using a laser printer (Figure 1). The plasma spots were dried at room temperature overnight. Each filter paper sheet was transferred to a separate zip-locked plastic bag with two small silica gel humidity absorber sachets and sent with a courier carrier to the Department of Forensic Sciences of Oslo University Hospital (Oslo, Norway) for analysis. The filter paper sheets were kept frozen until they were analysed. Frozen LPSs were sent with a courier carrier to the Division of Clinical Pharmacology, Groote Schuur Hospital, University of Cape Town (Cape Town, South Africa) for analysis. Ice packs were placed in the shipping container with the samples. Filter paper sheets spotted with 15 μl real plasma samples, blank plasma, calibrators and quality controls were cut out using scissors and transferred to a glass vial, and 25 μl of internal standard and 1 ml of methanol were added. The tubes were sonicated for 15 min and centrifuged at 3,500 rpm for 5 min. The extract was brought to dryness at 50°C under a flow of nitrogen and redissolved in 100 μl UHPLC mobile phase (10 mM ammonium formate buffer pH 3.1). The following substances were used as internal standards: 2H5-alprazolam, 13C6-amphetamine, 13C6-benzoylecgonine, 2H4-buprenorphine, 13C6-clonazepam, 13C6-cocaine, 13C2-codeine, 13C6-diazepam, 2H4-diclazepam, 2H5-fentanyl, 2H7-flunitrazepam, 13C6-MDMA, 13C6-methadone, 13C6-methamphetamine, 2H3-morphine, 13C6-nitrazepam, 13C6-oxazepam, 2H3-oxycodone,13C4-tetrahydrocannabinol, 13C-2H3-tramadol, 2H6-zolpidem and 2H8-zopiclone. The analysis was performed using an Acquity UPLC instrument coupled to a Xevo TQ-S MS/MS from Waters Corporation (Milford, MA, United States). Chromatographic separations were performed on a Kinetex biphenyl core-shell column (100 × 2.1 mm ID, 1.7 μm particles) from Phenomenex (Torrance, CA, United States) at a column temperature of 60°C, a flow rate of 0.5 ml/min and gradient elution with mobile phases consisting of 10 mM ammonium formate buffer pH 3.1 (solvent A) and MeOH (solvent B). The method has previously been validated for the analysis of blood samples.17 We used the recommended cut-off concentrations adjusted for differences in sample volume (Table 1). Drug findings above the cut-off concentrations were reported as positive. Recovery from DPSs was tested by comparing DPSs made with spiked plasma with blank DPSs spiked after extraction (n = 8). For each substance, two concentrations were used. Samples of 15 μl were applied onto a filter paper sheet and left to dry at room temperature. The dried sample spots were extracted and analysed as described in Section 2.3. Internal standards were added after extraction to correct for instrumental variations. Of each plasma sample, 100 μl was extracted with 600 μl acetonitrile and centrifuged for 5 min at 13,000 rpm. The supernatant was transferred to a 12 × 75 mm borosilicate glass tube and dried at 40°C under a flow of nitrogen. Once dried, each sample was reconstituted with solvent A containing 2H3-doxepin and 2H5-diazepam as internal standards. The analysis was performed using an HPLC Sciex Exion instrument coupled to an X500R Time-of-Flight Mass Spectrometer, all from Sciex (Framingham, MA, United States). The chromatographic separation was performed using a Kinetex 2.6 μm phenyl-hexyl column, 100A, 50 × 4.6 mm from Phenomenex, at a temperature of 30°C. Gradient elution was employed with 5 mM ammonium formate (solvent A) and 0.05% formic acid in methanol (solvent B). The estimated cut-off concentration was 20 ng/ml. Drugs identified using a library of more than 700 substances were reported as positive. The transportation of frozen plasma samples took about 24 h. When they arrived at the laboratory, the samples had thawed but were still cold. Samples were frozen until analysis. Analytical findings are presented in Table 1. Only tramadol, diazepam and benzoylecgonine were detected in the samples. For tramadol and diazepam, qualitative analysis of LPSs using HPLC-TOF-MS confirmed the presence of drugs in almost all DPSs that tested positive. However, qualitative analyses found traces of benzoylecgonine in some more LPSs than the analysis of DPSs revealed. Benzoylecgonine is a metabolite of cocaine. None of the samples that tested positive for benzoylecgonine (DPSs or LPSs) contained cocaine. Diazepam is a sedative drug that is often given to trauma patients during treatment. Tramadol is an opiate used for severe pain but is also commonly used for recreational purposes in parts of the world, including Cameroon and other African countries. We were unable to collect data on drug administration to the included trauma patients, so we do not know whether the concentrations represent therapeutic or recreational use, or a combination. Only 15 μl plasma was applied when preparing DPSs, and only one DPS spot was analysed. The cut-off concentration could be lowered if two or more spots were extracted together. The recovery from DPSs is presented in Table 2. The recovery rates were relatively low for amphetamines and MDMA (7–11%), zopiclone (10–13%) and for tramadol (12–13%) but higher for other substances. Extraction with methanol was chosen based on previously published methods for extracting drugs from dried blood spots.18, 19 Preliminary experiments revealed that extracting drugs from DPS at the required low concentration was challenging but methanol gave relatively good results. Good recovery for THC, which often can be found in much lower concentrations than for instance amphetamine, was considered a key factor. Differences between the two methods might be caused by different cut-off concentrations, low extraction recovery for some drugs when analysing DPSs and degradation during storage. Also, cross-contamination between samples on the filter paper might be an issue. However, it is an accepted practice to place multiple dried blood samples in the same container along with sachets of desiccant for shipment.20 It is therefore unlikely that cross-contamination of dry material would be a problem on sheets with multiple DPSs as the drugs to a large extent are trapped within the dried matrix of plasma proteins. A low degree of cross-contamination was confirmed as the analysis of DPSs found diazepam in only 1 out of 98 samples that were found to be negative for diazepam in LPSs and tramadol in 2 out of 57 found negative in LPSs. The numbers of positive drug findings in LPSs among samples that were negative when analysing DPSs were similar. Thus, the differences between methods are most likely caused by differences in cut-off concentrations of the two methods. The analysis were performed using test methods that had not been fully validated for the quantitation of drugs. Recovery was sufficient to detect drugs in relevant concentrations, although the low recovery for some of the analytes will give a higher detection limit. Other extraction methods should be explored in further work as well as a full validation. The transportation of frozen LPSs from Cameroon to South Africa was done in the presence of cold packs. The use of dry ice would reduce the probability of thawing samples during transportation. However, it was impossible to find a courier transporter that could transport samples with dry ice. This study shows that the use of large pre-printed sheets of filter paper is a promising method that can be used to collect DPSs for simple and cheap transportation of plasma samples to the analytical laboratory. Its use should therefore be further investigated. We would like to thank Alicia Evans at the Mass Spectrometry Laboratory, Division of Clinical Pharmacology, Groote Schuur Hospital, University of Cape Town, South Africa, for analysing liquid plasma samples with TOF mass spectrometry and Sandrine H. N. Nguemeche and Pulcherie J. B. Etoga at the Laboratoire Modern de Reference, Yaoundé, for their assistance in the DPS preparation. We also thank Ago Mrsa and Leon Reubsaeth at the Department of Pharmacy, University of Oslo, for providing the filter paper. The recruitment of patients and collection of plasma samples were financially supported by the International Council on Alcohol, Drugs and Traffic Safety. The authors have no conflict of interest to declare. Research data are not shared.

Source: Drug Testing and Analysis

DOI: 10.1002/dta.3545