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Screening of Antihistamine Agents (Diphenhydramine) with Blood and Urine Samples by REMEDi-HS® System
Ohtsuji M, Ohshima T, Takayasu T, Nishigami J, Kondo T, Lin Z, Minamino T
Department of Legal Medicine, Kanazawa University School of Medicine, Takara-machi 13-1, Kanazawa, 920, Japan
Diphenhydramine (Dip) is one of the antihistamine agents (Anti-His) which are often compounded in cold remedies in Japan. Because of its sedative effects, it is advisable for drivers not to take it. However, as cold remedies can be obtained without a doctor's prescription, Dip is frequently taken by all age groups in Japan. We sometimes experience the traffic accidents' autopsy cases in which the urine specimens of drivers were Anti-His positive. The noninvasive screening for Anti-His on drivers as well as alcohol seems, therefore, necessary in order to examine whether etiologically Anti-His was directly involved in the occurrence of traffic accident or not.
Urine samples were collected from 5 male volunteers at 0, 1, 2, 3, 6, 9, 24, 36 and 50 hours after Dip intake (a single 30 or 60mg oral dose), and 1 ml of each urine sample was used for the analysis by REMEDi-HS® based on HPLC system. Dip and its 3 different kinds of metabolites, which reached at their maximum concentrations at 3 hours, were detected until at 24 to 50 hours. Maximum concentration of Dip ranged from 0.53 to 4.15 µg/ml (mean value = 1.93 µg/ml. SD= 1.53). At 1 hour though Dip was often undetected probably because of its very low concentration, one specific metabolite, on the other hand, was detected in the urine.
These results show that REMEDi-HS® is useful for the Dip screening in human urine samples and that the metabolite is an early index suitable for revealing Dip intake.
Diphenhydramine is one of the antihistamine agents which are often compounded in antiphlogistics and in cold remedies in Japan. Because of its sedative effects, it is advisable for automobile drivers not to take it. However, diphenhydramine can be obtained without a doctor's prescription, it is easily taken by all age groups, nevertheless before driving, in Japan. The traffic accidents' autopsy cases, in which the serum and/or urine samples of drivers were antihistamine agents positive, have been sometimes experienced. Therefore, the non-invasive and/or very slightly invasive screening for antihistamine agents as well as alcohol seems to be important in order to examine to what extent antihistamine agent was etiologically involved in the occurrence of traffic accident. Screening of antihistamine agent (diphenhydramine) with blood and urine samples by REMEDi-HS® system (BIO-RAD, Hercules, CA, USA), which has recently begun to be used in Japan, is performed, and the results were compared with those by gas chromatography-mass spectrometry (GC-MS) method.
MATERIALS AND METHODS
Blood and urine samples were collected from the 17 male volunteers (from 18 to 27 in age) at 0, 1, 2, 3, 6, 9 and 24 hours after the oral ingestion of 3 to 12 pills (10 mg diphenhydramine hydrochloride was included in a pill). Urine samples were, moreover, collected at 36 and 48 hours. Each blood sample was centrifuged 3000 revolutions per minute (rpm) for 5 minutes (min), and the serum was extracted. These serum and urine samples were then analyzed by REMEDi-HS system. Before each sample collection every volunteer's consent was obtained in writing.
REMEDi-HS consists of high performance liquid chromatography (HPLC) system with computer and scanning ultraviolet (UV) detector. It is able to analyze about 500 kinds of basic and neutral drugs for about 20 min by the same analytical procedure. Four cartridges are used in this system; two cartridges initially separate drugs from proteins, salts and so on, and the other two cartridges produce an analytical differentiation of drugs, based on their characteristic retention times. Sample preparing procedures for this system were as follows; one ml serum or urine samples were mixed with 0.2 ml internal standard solution (IS) in a microfuge tube and were centrifuged 9500 rpm for 1 min. Serum samples were, moreover, centrifuged at 5000 rpm for 5 min in the centrifugal filter which was for REMEDi-HS's exclusive use. And then they were placed in the automated sampler. After necessary information was inputted, the analyzing can be started. IS contains 2 µg/ml N-ethyl-nordiazepam and 3 µg/ml chlorpheniramine and is for exclusive use of REMEDi-HS.
Moreover, to evaluate the quantitation value measured by REMEDi-HS, randomly selected samples were also analyzed by GC-MS. Extraction procedure for GC-MS analysis was as follows; two µg chlorpheniramine maleate was added as IS to 1 ml serum or urine samples. The samples were alkalized to pH 9.0 with 0.2 M NaOH. Thereafter, the samples were applied to an Extrelut column® and eluted with 20ml mixed solution of dichloromethane and isopropyl alcohol in the ratio of 85:15 (volume). The eluent was evaporated to dryness by nitrogen gas and the residue was reconstituted in 40 µl ethanol. One µl of the ethanol solution was analyzed by GC-MS. The conditions of GC-MS were as follows; A QP-1000 instrument (Shimadzu, Japan) equipped with a DB-1 column (15 m x 0.53 mm i.d., 1.5 µm film thickness, J & W Scientific, Folsom, CA, USA) was used. The column temperature was programmed from 180°C to 250°C with the increase at 10°C/min. The injection temperature was at 250°C, and helium flow rate was 15 ml/min. The mass spectrometer was operated in the positive electron impact mode by selected ion monitoring. The ionization energy and emission current were 70eV and 60µA, respectively.
Serum sample; an 18-year-old male who ingested orally 100 mg of diphenhydramine hydrochloride.
Urine sample; a 27-year-old male who ingested orally 120 mg of diphenhydramine hydrochloride.
dip., diphenhydramine. N-des., N-desmethyldiphenhydramine.
* diphenhydramine metabolite. IS1, N-ethylnordiazepam.
Diphenhydramine and N-desmethyldiphenhydramine began to be detectable by REMEDi-HS system in the serum samples 1 or 2 hours after the drug ingestion, and other two metabolites as well as them were detected in the urine samples (Figure 1). N-desmethyldiphenhydramine was detected in 50 samples out of 66 diphenhydramine positive serum samples, and diphen-hydramine metabolites were detected in every diphenhydramine positive urine sample (104 samples). Moreover, diphenhydramine metabolites were detected in 20 diphenhydramine negative urine samples. The detection of its metabolites in addition to diphenhydramine confirmed the intake of the drug.
The curves showed excellent linearity in the range of 0.04 µg/ml to 3 µg/ml, and the lower detection limit of diphenhydramine by this system was 0.04 µg/ml in the both samples.
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