شیمى کاربردى روز

شیمى کاربردى روز

کمی سازی پیشرفته اسیدهای آمینه هم‌شویش با استفاده از تحلیل تصویر مبتنی بر کروماتوگرافی لایه نازک و کالیبراسیون چند متغیره

نوع مقاله : مقاله علمی پژوهشی

نویسندگان
1 گروه شیمی، دانشکده علوم پایه، دانشگاه سمنان، سمنان، ایران
2 گروه شیمی، دانشکده علوم، دانشگاه محقق اردبیلی، کد پستی 56199-11367، اردبیل، ایران و گروه علوم محیطی، دانشکده علوم، دانشگاه زنجان، کد پستی 45371-38791، زنجان، ایران
3 مرکز تحقیقات سلامت محیط کار، دانشگاه علوم پزشکی نیشابور، نیشابور، ایران
چکیده
ل-گلوتامین (Gln) و گلیسین (Gly) دو اسید آمینه با زمان‌های بازداری دارای همپوشانی در کروماتوگرافی لایه نازک (TLC) هستند. این همپوشانی، چالش تحلیلی قابل توجهی را ایجاد می‌کند، به خصوص زمانی که هر دو ترکیب در یک مخلوط وجود داشته باشند، زیرا ممکن است نقاط آنها روی صفحه TLC به طور کامل تفکیک نشوند. در اینجا، ما از آنالیز تصویر مبتنی بر تلفن هوشمند همراه با رگرسیون حداقل مربعات جزئی (PLS) برای تعیین مقدار Gln/Gly نیمه جدا در TLC استفاده کردیم. یک تلفن هوشمند تصاویر صفحات TLC را تحت شرایط نوری ثابت ثبت کرد و نرم‌افزار سفارشی آنها را برای تولید پروفایل‌های کروماتوگرافی پردازش کرد. منحنی‌های کالیبراسیون پاسخ‌های خطی، با محدودیت‌های تشخیص 0.007 مولار و 0.008 مولار به ترتیب برای Gln و Gly را نشان دادند. با وجود بهینه‌سازی فاز متحرک، تفکیک کامل نقطه‌ای غیرقابل دستیابی بود. برای رسیدگی به این موضوع، رگرسیون حداقل مربعات (PLS) سیگنال‌های ادغام شده را تجزیه و تحلیل کرد و امکان تعیین مقدار دقیق را فراهم کرد. این روش با استفاده از یک محصول دارویی اعتبارسنجی شد ودرصد بازیابی %4/1 ± 4/100 برای Gln و % 1.0± 6/100 برای Gly به دست آمد که نشان‌دهنده قابلیت اطمینان آن برای نمونه‌های پیچیده است.
کلیدواژه‌ها
موضوعات

عنوان مقاله English

Enhanced Quantification of Co-eluting Amino Acids Using TLC based Image Analysis and Multivariate Calibration

نویسندگان English

Mehrdokht Hosseini Haghighi 1
Maryam Rajabi 1
Maryam Khoshkam 2
Toktam Mohammadi Moghaddam 3
1 Department of Chemistry, Faculty of Basic Sciences, Semnan University, Semnan, Iran
2 Department of Chemistry, Faculty of Sciences, University of Mohaghegh Ardabili,56199-11367, Ardabil, Iran & Department of Environmental Science, Faculty of Science, University of Zanjan, 45371-38791, Zanjan, Iran
3 Workplace Health Research Center, Neyshabour University of Medical Sciences, Neyshabour, Iran
چکیده English

L-Glutamine (Gln) and Glycine (Gly) are two amino acids with overlapping retention times in thin-layer chromatography (TLC). This overlap presents a significant analytical challenge, particularly when both compounds are present in the same mixture, as their spots on the TLC plate may not be fully resolved. Here, we used smartphone-based image analysis combined with PLS regression for semi-separated Gln/Gly quantification in TLC. A smartphone captured TLC plates images under consistent lighting conditions, and custom software processed them to generate chromatographic profiles. Calibration curves showed linear responses, with detection limits of 0.007 M and 0.008 M for Gln and Gly, respectively. Despite mobile-phase optimization, complete spot resolution was unachievable. To address this, partial least squares (PLS) regression deconvoluted the merged signals, enabling accurate quantification. The method was validated using a pharmaceutical product, yielding recovery rates of 100.4 ± 1.4% for Gln and 100.6 ± 1.0% for Gly, demonstrating its reliability for complex samples.

کلیدواژه‌ها English

TLC based image analysis
Multivariate calibration
Co-eluting components
Amino acids
[1] Teo, M., Khoo, L. W., & Chew, W. (2024). A simplified small-scale workflow for determination of complete protein-bound amino acids using pre-column derivatization HPLC method. Journal of Food Composition and Analysis135, 106571.
[2] Zhang, J., Yu, X., Yang, R., Zheng, B., Zhang, Y., & Zhang, F. (2024). Quality evaluation of Lonicerae Japonicae Flos from different origins based on high‐performance liquid chromatography (HPLC) fingerprinting and multicomponent quantitative analysis combined with chemical pattern recognition. Phytochemical Analysis35(4), 647-663.
[3] de Sousa Fontes, V. M., Pimentel, T. C., da Silva, A. B. M., Madruga, M. S., Magnani, M., & dos Santos Lima, M. (2024). An improved method for determining free amino acids by RP-HPLC/DAD with o-phthalaldehyde derivatization: Method evaluation in beers and wines. Food Chemistry435, 137591.
[4] Ichihara, K. I., Kohsaka, C., & Yamamoto, Y. (2021). Determination of proteinaceous free amino acids by gas chromatography. Analytical Biochemistry633, 114423.
[5] Posma, R. A., Bakker, S. J., Nijsten, M. W., Touw, D. J., & Tsikas, D. (2024). Comprehensive GC-MS Measurement of Amino Acids, Metabolites, and Malondialdehyde in Metformin-Associated Lactic Acidosis at Admission and during Renal Replacement Treatment. Journal of Clinical Medicine13(13), 3692.
[6] Ferreira, C. P., Antunes, F. T. T., Rebelo, I. N., da Silva Junior, C. A., Vilanova, F. N., Corrêa, D. S., & de Souza, A. H. (2020). Application of the UV–vis spectrophotometry method for the determination of glutamate in the cerebrospinal fluid of rats. Journal of Pharmaceutical and Biomedical Analysis186, 113290.
[7] Huang, C., & Kalodimos, C. G. (2017). Structures of large protein complexes determined by nuclear magnetic resonance spectroscopy. Annual review of biophysics46, 317-336.
[8] Nord, L. I., Vaag, P., & Duus, J. Ø. (2004). Quantification of organic and amino acids in beer by 1H NMR spectroscopy. Analytical chemistry76(16), 4790-4798.
[9] Soga, T., Kakazu, Y., Robert, M., Tomita, M., & Nishioka, T. (2004). Qualitative and quantitative analysis of amino acids by capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry. Electrophoresis25(13), 1964-1972.
[10] Wilson, S. F., James, C. A., Zhu, X., Davis, M. T., & Rose, M. J. (2011). Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC–MS/MS. Journal of pharmaceutical and biomedical analysis56(2), 315-323.
[11] Hasani, M., Yaghoubi, L., & Abdollahi, H. (2007). A kinetic spectrophotometric method for simultaneous determination of glycine and lysine by artificial neural networks. Analytical Biochemistry365(1), 74-81.
[12] Rajurkar, N. S., & Gaikwad, K. N. (2014). Identification and quantification of amino acids from medicinally important plants by using high-performance thin-layer chromatography. Journal of Liquid Chromatography & Related Technologies37(15), 2197-2205.
[13] Hodisan, T., Culea, M., Cimpoiu, C., & Cot, A. (1998). Separation, identification and quantitative determination of free amino acids from plant extracts. Journal of Pharmaceutical and Biomedical Analysis18(3), 319-323.
[14] Martens, J., & Bhushan, R. (1989). TLC enantiomeric separation of amino acids. International Journal of Peptide and Protein Research34(6), 433-444.
[15] Qiu, T., Li, H., & Cao, Y. (2010). Pre-staining thin layer chromatography method for amino acid detection. African Journal of Biotechnology9(50), 8679-8681.
[16] Sardesai, V. M., & Provido, H. S. (1970). The determination of glycine in biological fluids. Clinica chimica acta29(1), 67-71.
[17] Botrè, F., Botrè, C., Lorenti, G., Mazzei, F., Porcelli, F., & Scibona, G. (1993). Determination of L-glutamate and L-glutamine in pharmaceutical formulations by amperometric L-glutamate oxidase based enzyme sensors. Journal of pharmaceutical and biomedical analysis11(8), 679-686.
[18] Machado, T. R., & Fonseca, A. (2019). A new alternative method for quantitative analysis by thin layer chromatography using a lab-made multi-track device and image-based chromatograms. Microchemical Journal147, 127-132.
[19] Hemmateenejad, B., Farzam, S. F., & Mobaraki, N. (2014). Simultaneous measurement of leucine and isoleucine by multivariate image analysis-thin layer chromatography (MIA-TLC). Journal of the Iranian Chemical Society11(6), 1609-1617.
[20] Hemmateenejad, B., Mobaraki, N., Shakerizadeh-Shirazi, F., & Miri, R. (2010). Multivariate image analysis-thin layer chromatography (MIA-TLC) for simultaneous determination of co-eluting components. Analyst135(7), 1747-1758.
[21] Bedia, C., Sierra, A., & Tauler, R. (2021). Multimodal multisample spectroscopic imaging analysis of tumor tissues using multivariate curve resolution. Chemometrics and Intelligent Laboratory Systems215, 104366.
[22] Dhanapal, L., & Erkinbaev, C. (2024). Portable hyperspectral imaging coupled with multivariate analysis for real-time prediction of plant-based meat analogues quality. Journal of Food Composition and Analysis126, 105840.
[23] Brereton, R. G. (2003). Chemometrics: data analysis for the laboratory and chemical plant. John Wiley & Sons.
[24] Li, H., Qiu, T., Cao, Y., Yang, J., & Huang, Z. (2009). Pre-staining paper chromatography method for quantification of γ-aminobutyric acid. Journal of chromatography A1216(25), 5057-5060.
[25]  Li, Q. Z., Huang, Q. X., Li, S. C., Yang, M. Z., & Rao, B. (2012). Simultaneous determination of glutamate, glycine, and alanine in human plasma using precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography. The Korean journal of physiology & pharmacology16(5), 355-360.
[26] Lu, M. J., Chiu, T. C., Chang, P. L., Ho, H. T., & Chang, H. T. (2005). Determination of glycine, glutamine, glutamate, and γ-aminobutyric acid in cerebrospinal fluids by capillary electrophoresis with light-emitting diode-induced fluorescence detection. Analytica chimica acta538(1-2), 143-150.
[27] Piepponen, T. P., & Skujins, A. (2001). Rapid and sensitive step gradient assays of glutamate, glycine, taurine and γ-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis samples. Journal of Chromatography B: Biomedical Sciences and Applications757(2), 277-283.
[28] Hasani, M., Yaghoubi, L., & Abdollahi, H. (2007). A kinetic spectrophotometric method for simultaneous determination of glycine and lysine by artificial neural networks. Analytical Biochemistry365(1), 74-81.
[29] Pye, I. F., Stonier, C., & McGale, E. H. (1978). Double-enzymic assay for determination of glutamine and glutamic acids in cerebrospinal fluid and plasma. Analytical Chemistry50(7), 951-953.