Development and Validation of an Analytical Method for Glucuronolactone in Energy Drinks by Hydrophilic Interaction Liquid Chromatography-electrospray Tandem Mass Spectrometry원문보기
에너지 음료는 카페인을 주성분으로 타우린, 비타민 같은 다른 energy-enhancing 성분을 함유하고 있다. 미국과 유럽에서는 글루쿠로노락톤이 에너지 음료에 첨가될수 있으나, 국내에서 의약품으로는 허가되어 있다. 따라서 식품 첨가물로는 그 사용이 금지 되어 있어, 지속적으로 수입 및 유통 음료에서 시험검사를 하여 규제하고 있다. 현재 분석법으로 사용하는 LC-PDA 법은 복잡한 유도체화 과정을 거치고, 음료 중에 당류들이 위양성 결과를 나타내기도 한다. 이런 기존 방법의 단점을 개선하기 위해 HILIC-ESI-MS/MS(hydrophilicinteraction liquid chromatography coupled to electrospray ionization tandem mass spectrometry)를 이용한 분석법을 개발하고, 선택성, 직선성, 검출한계, 정량한계, 정밀도, 정확성, 재현성에 대하여 분석법 유효성 검증을 수행했고, AOAC, EURACHEM 가이드라인에 부합되는 결과를 얻었다.
에너지 음료는 카페인을 주성분으로 타우린, 비타민 같은 다른 energy-enhancing 성분을 함유하고 있다. 미국과 유럽에서는 글루쿠로노락톤이 에너지 음료에 첨가될수 있으나, 국내에서 의약품으로는 허가되어 있다. 따라서 식품 첨가물로는 그 사용이 금지 되어 있어, 지속적으로 수입 및 유통 음료에서 시험검사를 하여 규제하고 있다. 현재 분석법으로 사용하는 LC-PDA 법은 복잡한 유도체화 과정을 거치고, 음료 중에 당류들이 위양성 결과를 나타내기도 한다. 이런 기존 방법의 단점을 개선하기 위해 HILIC-ESI-MS/MS(hydrophilic interaction liquid chromatography coupled to electrospray ionization tandem mass spectrometry)를 이용한 분석법을 개발하고, 선택성, 직선성, 검출한계, 정량한계, 정밀도, 정확성, 재현성에 대하여 분석법 유효성 검증을 수행했고, AOAC, EURACHEM 가이드라인에 부합되는 결과를 얻었다.
A rapid, sensitive analytical method for glucuronolactone in beverages was developed and validated using hydrophilic interaction liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). To determine the optimum analytical conditions for glucuronolactone, t...
A rapid, sensitive analytical method for glucuronolactone in beverages was developed and validated using hydrophilic interaction liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). To determine the optimum analytical conditions for glucuronolactone, three different kinds of HILIC columns and two mobile phases with different pH values were examined. An amide-bonded stationary phase with a pH 9 acetonitrile-rich mobile phase was the best condition in terms of column retention, ESI-MS/MS response area, and signal-to-noise ratio. After extraction, glucuronolactone was separated through the HILIC amide column and detected by negative ESI-MS/MS in selected reaction monitoring (SRM) mode. Nine energy drinks sold in Korea were spiked with glucuronolactone at a concentration of 5 ng/mL; the Monster $Energy^{TM}$ sample showed the smallest peak area and its signal-to-noise ratio was used for method validation. Good linearity was obtained in the concentration range from 20 to 1500 ng/mL with a correlation coefficient > 0.998. The developed method had a limit of detection (LOD) of 6 ng/mL and a limit of quantitation (LOQ) of 20 ng/mL. The recovery of this method at concentration of 20, 100, 500, and 1000 ng/mL was 96.3%-99.2% with relative standard deviations (RSD) of 1.6%-14.0%. A reproducibility precision assessment at concentration of 100 and 500 ng/mL was carried out among three laboratories. The recovery of that evaluation was 95.1%-102.3% with RSD of 2.7%-7.0%. An analysis of variance indicated that there was no difference between the recovery results of the three laboratories at the 5% significance level. The validated method is applicable to inspecting beverages adulterated with glucuronolactone in Korea.
A rapid, sensitive analytical method for glucuronolactone in beverages was developed and validated using hydrophilic interaction liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). To determine the optimum analytical conditions for glucuronolactone, three different kinds of HILIC columns and two mobile phases with different pH values were examined. An amide-bonded stationary phase with a pH 9 acetonitrile-rich mobile phase was the best condition in terms of column retention, ESI-MS/MS response area, and signal-to-noise ratio. After extraction, glucuronolactone was separated through the HILIC amide column and detected by negative ESI-MS/MS in selected reaction monitoring (SRM) mode. Nine energy drinks sold in Korea were spiked with glucuronolactone at a concentration of 5 ng/mL; the Monster $Energy^{TM}$ sample showed the smallest peak area and its signal-to-noise ratio was used for method validation. Good linearity was obtained in the concentration range from 20 to 1500 ng/mL with a correlation coefficient > 0.998. The developed method had a limit of detection (LOD) of 6 ng/mL and a limit of quantitation (LOQ) of 20 ng/mL. The recovery of this method at concentration of 20, 100, 500, and 1000 ng/mL was 96.3%-99.2% with relative standard deviations (RSD) of 1.6%-14.0%. A reproducibility precision assessment at concentration of 100 and 500 ng/mL was carried out among three laboratories. The recovery of that evaluation was 95.1%-102.3% with RSD of 2.7%-7.0%. An analysis of variance indicated that there was no difference between the recovery results of the three laboratories at the 5% significance level. The validated method is applicable to inspecting beverages adulterated with glucuronolactone in Korea.
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제안 방법
The recovery results were also used to determine the intra- and inter-day precision, which were calculated as RSD values. A reproducibility precision experiment was conducted among three laboratories at two concentration levels (100 and 500 ng/mL), and this data underwent analysis of variance to identify whether there were any differences among the three laboratories.
The LOD and LOQ determined from the S/N are considered to have somewhat lower reliability than those obtained from the other two methods because the algorithms used to calculate S/N differ according to the analysis instruments and computing software. After examining the LOD and LOQ values obtained using the three methods, the LOD and LOQ of the method were decided as 6 ng/mL and 20 ng/mL respectively.
Three HILIC columns with different column chemistries, including amide, amino, and non-derivatized silica, and two types of eluents, including an acidic (pH 3) and alkaline (pH 9) eluent, were tested to determine the optimized LC-MS/MS analytical conditions. Among the various combinations of columns and eluents, an amide column and alkaline acetonitrile-rich mobile phase performed best for evaluating the peak signal-to-noise ratio, column retention, and ESI-MS response. Considering the guidelines of international organizations related to chemical analysis method validation, such as AOAC (Associaion of Analytical Communities), EURACHEM (Analytical Chemistry in Europe), and ICH (International Council for Harmonisation), the developed method was fully validated according to specificity, linearity, LOD, LOQ, precision, and accuracy.
For accuracy and precision validation, the recovery was evaluated at four concentration levels (20, 100, 500, and 1000 ng/mL) in quintuplicate for three days. The recoveries at the four concentration levels ranged from 96.
The LOD and LOQ values were determined three ways: from the signal-to-noise ratios (S/N)10-11), from the standard deviation of the calibration curve intercept and the average calibration curve slope12), and from the standard deviation obtained for the repetitive analysis of a concentration10-11). The linearity of the method was assessed using the correlation coefficient acquired from the calibration data in the range of 20-1500 ng/mL using a linear regression model, and changes in the linearity were observed over three days. The accuracy was evaluated as the recovery of known amounts of glucuronolactone because a reference material could not be obtained13).
The recovery was performed at four concentration levels (20, 100, 500, and 1000 ng/mL) using a matrix-matched calibration. The recovery experiment was performed in quintuplicate for three days. The recovery results were also used to determine the intra- and inter-day precision, which were calculated as RSD values.
The accuracy was evaluated as the recovery of known amounts of glucuronolactone because a reference material could not be obtained13). The recovery was performed at four concentration levels (20, 100, 500, and 1000 ng/mL) using a matrix-matched calibration. The recovery experiment was performed in quintuplicate for three days.
Nine energy drinks sold at a local market were spiked with glucuronolactone at 5 ng/mL to select a blank sample for method validation. The specificity of the method was evaluated by comparing the chromatograms of the blank and spiked samples. The LOD and LOQ values were determined three ways: from the signal-to-noise ratios (S/N)10-11), from the standard deviation of the calibration curve intercept and the average calibration curve slope12), and from the standard deviation obtained for the repetitive analysis of a concentration10-11).
Using the HILIC-ESI-MS technique, glucuronolactone in beverages was analysed in a rapid and simple way. Three HILIC columns with different column chemistries, including amide, amino, and non-derivatized silica, and two types of eluents, including an acidic (pH 3) and alkaline (pH 9) eluent, were tested to determine the optimized LC-MS/MS analytical conditions. Among the various combinations of columns and eluents, an amide column and alkaline acetonitrile-rich mobile phase performed best for evaluating the peak signal-to-noise ratio, column retention, and ESI-MS response.
To increase the relevancy and applicability of the analytical method, the sample with the lowest MS response among the spiked samples was employed as the matrix blank sample for the method validation.
Using the established tandem mass spectrometry conditions, the combinations of three different HILIC columns (AtlantisTM Silica, XbridgeTM BEH Amide, and UnisonTM UK-Amino) and two types of eluents (pH 3 and pH 9 mobile phases) were evaluated at a concentration of 100 ng/mL spiked to determine the optimized LC analytical conditions. The LC was operated in isocratic elution mode at a flow rate of 0.
데이터처리
The linearity of the method was assessed by examining the correlation coefficients of the calibration curves. The matrix blank sample was spiked with glucuronolactone at concentration levels of 20, 50, 100, 200, 500, 1000, and 1500 ng/mL.
The recovery experiment was performed in quintuplicate for three days. The recovery results were also used to determine the intra- and inter-day precision, which were calculated as RSD values. A reproducibility precision experiment was conducted among three laboratories at two concentration levels (100 and 500 ng/mL), and this data underwent analysis of variance to identify whether there were any differences among the three laboratories.
참고문헌 (14)
Heckman, M.A., Sherry, K., Gonzalez, De., Mejia. E.: Energy drinks: An assessment of their market size, consumer demographics, ingredient profile, functionality, and regulations in the United States, Compr. Rev. Food Sci. Food Saf., 9, 303-317 (2010).
Seifert, S.M., Schaechter, J.L., Hershorin, E.R,. Lipshultz, S.E.: Health effects of energy drinks on children, adolescents, and young adults, Pediatrics, 127, 511-528 (2011).
Suzuki, S., Hayase, S., Nakano, M., Oda, Y., Kakehi, K.: Analysis of glucuronolactone and glucuronic acid in drug formulations by high-performance liquid chromatography. J. Chromatogr. Sci., 36, 357-360 (1998).
Grumbach, E.S., Fountain, K.J.: Comprehensive Guide to HILIC. Waters Corporation: Milford MA (2010).
Schlichtherle-Cerny, H., Affolter, M., Cerny, C.: Hydrophilic interaction liquid chromatography coupled to electrospray mass spectrometry of small polar compounds in food analysis. Anal. Chem., 75, 2349-2354 (2003).
Ricciutelli, M., Caprioli, G., Cortese, M., Lombardozzi, A., Strano, M., Vittori, S., Sagratini, G.: Simultaneous determination of taurine, glucuronolactone and glucuronic acid in energy drinks by ultra high performance liquid chromatography-tandem mass spectrometry (triple quadrupole). J. Chromatogr. A., 1364, 303-307 (2014).
AOAC. Guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals. AOAC (2002).
International Conference on Harmonization (ICH). Validation of analytical procedures: Text and methodology Q2 (R1). ICH (1994).
U.S. Department of Health and Human Services, Food and Drug Administration, Center for Veterinary Medicine. 1999. Validation of analytical procedures: Methodology.
EURACHEM. The fitness for purpose of analytical methods: A laboratory guide to method validation and related topics. EURACHEM (1998).
Shabir, G.A.: Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J. Chromatogr. A., 987, 57-66 (2003).
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