Pseudoephedrine과 유도체화 시약의 TLC상 직접반응과 이미지 분석을 통한 검출한계 및 반정량분석법에 관한 선행평가
Preliminary Evaluation of Detection Limit and Semi-quantitative Analysis Method for Pseudoephedrine Using in situ Pre-chromatographic Reaction with Derivatization Reagents and Image Analysis on TLC
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초록
Pseudoephedrine (PSE)은 methamphetamine의 불법합성에 중요한 화학적 전구체 중의 하나로 이에 대한 신속하고 용이한 동정과 반정량을 목적으로 하는 방법의 개발을 위한 예비실험을 실행하였다.TLC상에서 PSE와 유도체화 시약을 직접적(in situ, co-spot)으로 반응시켜서 생성된 유도체를 확인하여 검출한계를 결정하고 이들에 대한 이미지를 이미지 분석 프로그램으로 재현성과 직진성에 대해 분석하 여 반정량적인 분석에 적합한지 평가하였다. PSE의 칼람 분리 전(pre-column)에 행하는 직접적인(co-spot) 유도체화 반응성을 평가하고자 PSE를 실리카 젤(SiO2) TLC 상에서 3종의 검출특징이 다른 유도체화 시약 9-fluorenylmethyl chloroformate (FMOC-Cl), dansyl chloride (Dansyl-Cl) 및 dabsyl chloride (Dabsyl-Cl)와 각 각 반응시켰다. 유도체화 반응 생성물을 UV 혹은 가시광선에서 육안으로 확인한 후, TLC 이미지 분석을 통하여 생성 spot(s)에 대한 반응의 재현성을 평가하고, 추가적으로 반 정량분석을 위한 직선성을 평가하였다. 모든 유도체화 시약들은 TLC상에서 PSE와 반응하여 가시광선 혹은 UV-active한 작용기를 함유한 유도체를 생성하였으며 각 시약에 따른 PSE와의 반응성의 차이가 관찰되었다. PSE의 검출한계 (Limit of detection, LOD, 2 μL per spot, mg/mL)농도는 FMOC-Cl과(상온에서 노출하여 10분간 반응), Dansyl-Cl 사용 시(반응 spots들을 유리판으로 덮은 상태에서 70 oC로 가열) 0.001~0.005 mg/mL의 농도 구간에서 양호하였으나 Dabsyl-Cl의 경우에는 가열을 하였음에도 0.25 mg/mL의 높은 농도에서 검출이 가능하였다. 반응의 재현성(RSD, 평균치, %)은 FMOC-Cl의 사용 시 PSE 0.5 mg/mL의 농도에서 2.14 ~ 2.27%로 양호하였으나 저 농도 0.1 mg/mL에서는 5.87 ~ 6.20%으로 변동이 심하였고, Dansyl-Cl의 경우 에는 두 가지 농도(0.5 및 0.05 mg/mL)에서 4.63 ~ 5.61%로 역시 변동이 심하였다. 반응의 직선성(linearity,R2) 평가에서 PSE의 높은 농도구간(0.1 ~ 1.0 mg/mL)에서는 FMOC-Cl과 Dansyl-Cl시약 모두 PSE의 농도에 따른 생성 유도체의 면적과의 상관관계가 0.96 ~ 0.98로 비교적 양호한 상관성을 보였으나, PSE의 검출한계 농도에서 1.0 mg/mL의 농도구간에서는 0.90 ~ 0.92로 감소하였다. 현재의 방법으로 세 가지의 유도체들이 다소 낮은 재현성으로 생성이 되었으며, PSE에대한 검출한계 농도는 유도체화 이전의 검출한계의 농도보다 160 ~ 1000배가량 낮추었으나, 본방법의 PSE에 대한 반정량적인 사용을 위한 방법으로서의 유효성을 보이기 위해서는 사용시약의 다양한 농도변화, 다른 반응용매 사용에 의한 영향, 반응온도의 변화 등에 대한 PSE의 다양한 농도에서의 유도체화 반응의 재현성을 높이기위한 추가적인 실험결과가 필요하다.
Abstract
Pseudoephedrine (PSE) is the critical chemical precursor involved in the illegal production ofmethamphetamine. A preliminary test for the rapid and easy identification and semi-quantitative determinationof PSE was performed. PSE derivatives generated from direct reaction (in situ ,co-spot) of PSE with derivatizing reagents were identified and used to determine the LOD. The TLC images were analyzed with densitometric image analysis programs to determine the repeatability and linearity of derivatization reaction for a potential semi-quantitative analysis. PSE was reacted in situ (co-spot) to evaluate their derivatization reactivity in the pre-column mode on a silica gel TLC plate with FMOC-Cl, Dansyl-Cl or Dabsyl-Cl as derivatizing reagents. The derivatized products were identified under naked eyes with or without UV light and the TLC image analysis of corresponding spots was performed using the densitometric image analysis for the semi-quantitative evaluation. All the derivatizing reagents generated compounds containing strongly visible or UV-absorbing FMOC-, Dansyl- and Dabsyl- groups on the TLC plate after reacting with PSE. However, a different reactivity of PSE with the reagents was observed. In the experiment using FMOC-Cl (reaction spots exposed with air at room temperature for 10 min.) and Dansyl-Cl (reaction spots covered with glass plate while heated at 70 oC for 10 min.), the LODs (Limit of detection, 2 μL/spot, mg/mL) of PSE was in the low concentration range of 0.001 ~ 0.005, but the higher LOD concentration of 0.25 was found when Dabsyl-Cl was used even when the reaction spots were covered and heated. The RSD value (average, %) to test the repeatability of the derivatization reaction for PSE (0.5 mg/mL) was in the range of 2.14 ~ 2.27% when FMOC-Cl used. But a high RSD value range of 5.87 ~ 6.20% was shown at the low concentration (0.1 mg/mL) of PSE. Also at two concentrations (0.5 mg/mL and 0.05 mg/mL) of PSE with Dansyl-Cl, the high RSD value range of 4.63 ~ 5.61% was found. In the test of the linearity (R2) of the derivatization reaction at a high concentration range (0.1 ~ 1.0 mg/mL) of PSE with FMOC-Cl and Dansyl-Cl, a relatively good correlation was shown to be in the range of 0.96 ~ 0.98 between the areas of spots of generated derivatives and concentration of PSE. However, the R2 values calculated from the concentration of the LOD of PSE up to a high concentration (1mg/mL) of PSE was dropped into the range of 0.90 ~ 0.92. The current method generated three derivatives with rather low repeatability and provided a way to improve the LOD of PSE to 160 ~ 1000 times compared to the TLC method of underivatized PSE. To demonstrate the validity of the current method to be used for a semi-quantitative analysis of PSE by showing the high repeatability of a derivatization reaction at various concentration of PSE, additional experimental results are required such as the various changes of the concentration of derivatization reagents, the usage of other reaction solvent, changes of reaction temperature, etc.
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