Ephedrine의 TLC상 직접적인 유도체화 반응 시 보조용매 DMF 사용을 통한 검출한계 증강 및 이미지 분석
Improvement of Detection Limit of Ephedrine using Co-solvent DMF for in situ Direct Derivatization Reaction and Image Analysis in TLC
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초록
Ephedrine (EP)은 교감신경 흥분성 아민으로 다수의 약물, 한약, 건강보조제 등에서 발견된다. EP는 pseudoephedrine (PSE)의 부분입체 이성질체로 두 화합물 모두 methamphetamine (ME)의 불법합성에 사용되어지며 특히 EP는 체중감량 및 경기력 향상을 위하여 오용 또는 남용되어지는 중요한 자극제이다. EP에 대한 빠르고 용이한 확인 및 반정량 방법으로 TLC상 직접반응(co-spot, in situ)법을 예비적으로 평가하였다. EP와 3종류의 유도체화 시약(9-fluorenylmethyl chloroformate (FMOC-Cl), dansyl chloride (Dansyl-Cl) 및 dabsyl chloride (Dabsyl-Cl))을 각각 보조용매인 N,N-dimethylformamide (DMF)의 존재(1 μL per spot)하에서 미사용 시와 비교 및 검출한계(Limit of Detection, LOD, 2 μL per spot, mg/mL)를 결정하였다. 또한 TLC이미지를 농도계(densitometric) 이미지분석 프로그램으로 분석하여 재현성 및 상관관계를 평가하였다. EP의 검출한계농도는 DMF의 존재 하에서 FMOC-Cl과(10분간 상온에서 노출하여 반응), Dansyl-Cl 사용 시(70 oC에서 반응 spots들을 유리판으로 덮은 상태에서 반응)에 각각 0.01, 0.05 mg/mL로 DMF를 사용하지 않은 경우보다 5 ~ 10배의 증강을 보였고, Dabsyl-Cl의 경우 약 100배의 증강이 관찰되었다. 이는 DMF가 유도체 형성 시 전이상태에서 수소결합을 통한 이탈기(-Cl)의 이탈촉진과 생성물(HCl)의 안정화에 따른 용매효과로 사료된다. FMOC-Cl의 사용 시 유도체화 반응의 재현성(RSD, 평균치, %)은 1.70 ~ 3.48%였으나 Dansyl-Cl의 경우에는 2.48 ~ 8.10%로 변동이 심하였다. EP의 농도에 대한 생성 유도체 면적과의 상관관계(linearity, R2) 평가에서는 좁은 EP의 농도구간(0.01 ~ 0.25 mg/mL)에서 FMOC-Cl사용 시 그리고 Dabsyl-Cl (EP, 0.01 ~ 0.5 mg/mL)사용에서 0.98 ~ 0.99로 비교적 양호한 상관성을 보였다. 현재의 방법이 EP에 대한 동정 및 반정량적인 분석을 위한 방법으로서의 유효성을 보이기 위해서는 반응의 재현성 증가시키기 위한 추가적인 실험결과가 요구된다.
Abstract
Ephedrine (EP) is a sympathomimetic amine and found in many pharmaceutical preparation, Chinese crude drugs and nutritional supplements. EP is a diastereomer of pseudoephedrine (PSE) and both have been used for the illegal production of methamphetamine (ME). Also EP is an important stimulant to be misused and abused for weight loss and performance enhancement. The rapid and easy identification and semi-quantitative analysis of EP using TLC co-spot method was performed as a preliminary step. A direct reaction (in situ, co-spot) of EP with three derivatizing reagents (FMOC-Cl, Dansyl-Cl or Dabsyl-Cl) along with/without co-solvent N,N-dimethylformamide (DMF) has been tested to determine the LOD of EP. Also the resultant TLC images of generated EP derivatives were analyzed with densitometric image analysis programs to test the repeatability and linearity of derivatization method. When the reagents FMOC-Cl (reaction spots at room temperature for 10 min.) and Dansyl-Cl (reaction spots covered with glass plate while heated at 70 oC for 10 min.) were reacted with EP along with co-solvent DMF, the LODs (Limits of detection, 2 μL/spot, mg/mL) of EP was improved about 5 and 10 times (0.01 ~ 0.05), respectively. But about 100 times of improvement was identified for Dabsyl-Cl. This can explained by dipolar aprotic solvent’s effects through forming hydrogen bonding with the elimination group (-Cl) for faster elimination and HCl by-product to form stable structure within the transition state. The RSD value (average, %) to test the repeatability was in the range of 1.70 ∼ 3.48% when FMOC-Cl used. A wider RSD value range of 2.48 ~ 8.10% was found with Dansyl-Cl. In the test of the co-relationship (linearity, R2) of the derivatization reaction at a narrow concentration range (0.01 ~ 0.25 mg/mL) of EP with FMOC-Cl, and with Dabsyl-Cl (EP, 0.01 ~ 0.5) a relatively good correlation was shown to be in the range of 0.98 ~ 0.99 between the areas of spots of generated derivatives and concentration of EP. To validate the current TLC method to be used at the scene or the forensic lab. to determine the LOD of EP and for a semi-quantitative determination of EP, the higher reaction repeatability of a derivatization reaction at various concentration of EP should be shown and additional TLC test results are required.
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