Malaysian Journal of Analytical Sciences, Vol 28 No 5 (2024): 1059 – 1070

 

DETECTION OF DIURETICS IN WHEY PROTEIN UTILIZING LIQUID-LIQUID EXTRACTION COUPLED WITH LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LLE-LC-MS/MS): A COMPREHENSIVE ANALYSIS

 

(Pengesanan Diuretik dalam Protin Whey Menggunakan Pengekstrakan Cecair-Cecair Berpasangan dengan Kromatografi Cecair - Spektrometri Jisim (LLE-LC-MS/MS): Satu Analisis Komprehensif)

 

Siti Khadijah Ab Rahman1*, Yeo Wee Kian1, and Umi Zulaikha Mohd Azmi2

 

1National Sport Institute, Kompleks Sukan Negara, Bukit Jalil, 57000 Kuala Lumpur, Malaysia

2Malaysian Nuclear Agency, 43000 Kajang, Selangor, Malaysia

 

*Corresponding author: khadijah@isn.gov.my

 

 

Received: 15 March 2024; Accepted: 10 July 2024; Published:  27 October 2024

 

 

 

Abstract

Protein supplements are widely consumed by athletes and recreationally active adults to enhance training adaptation, performance, and muscle recovery. However, the popularity of these supplements has led to instances of adulteration with banned substances, including diuretics. Diuretics, typically used for medical conditions like hypertension and edema, are illicitly added to supplements to promote weight loss and mask other doping agents. Their presence poses significant health risks, including electrolyte imbalances and renal dysfunction, and contributes to doping violations in sports. Despite the critical need to monitor diuretic contamination in protein supplements, there is a noticeable gap in the literature regarding optimized extraction methods for these compounds. This study aims to evaluate the efficiency of various solvents in extracting diuretic compounds from protein supplements. By assessing the extraction recovery rates and optimizing the selection of solvents extraction, this research seeks to enhance the sensitivity, specificity, and accuracy of liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. This study investigates the extraction efficiency and matrix effects of methanol and acetonitrile for detecting diuretics in whey protein using Liquid-Liquid Extraction (LLE) coupled with LC-MS/MS. Different solvent compositions 100%, 70%, and 50% methanol (100M, 70M, 50M) and acetonitrile (100A, 70A, 50A) were evaluated to optimize recovery rates and minimize matrix interference. Results show that methanol consistently outperformed acetonitrile in recovering diuretic compounds from whey protein. At 100M demonstrated the highest average recovery rates (49.639% to 99.735%) with moderate signal enhancement and minimal suppression effects, indicating effective mitigation of matrix interference. Similarly, 70M maintained balanced matrix effects and reliable recoveries (46.976% to 94.492%), making it a robust alternative for diuretic analysis. In contrast, acetonitrile exhibited greater variability in matrix effects and lower recovery rates. For instance, 100A showed significant signal suppression (0.070% to 9.267%), suggesting limitations in solubilizing diuretics from whey protein. While 70A provided a more stable profile, it still showed variability (matrix effects from -44.539% to 29.493%) compared to methanol. The study highlights the critical role of solvent selection in minimizing matrix effects and ensuring accurate diuretic quantification in complex food matrices. Methanol’s superior solvating power and polarity contribute to its effectiveness in mitigating matrix interference compared to acetonitrile. This research provides valuable insights for analytical chemists and food scientists aiming to improve the accuracy and consistency of diuretic analysis in food matrices, thereby ensuring consumer safety and regulatory compliance.

 

Keywords: diuretics, liquid-liquid microextraction, liquid chromatography, whey protein

 

Abstrak

Suplemen protin digunakan secara meluas oleh atlit dan individu yang aktif secara rekreasi untuk meningkatkan penyesuaian latihan, prestasi, dan pemulihan otot. Namun, populariti suplemen ini telah menyebabkan terjadinya pemalsuan dengan bahan terlarang, termasuk diuretik. Diuretik, yang biasanya digunakan untuk keadaan kesihatan seperti hipertensi dan edema, telah ditambah ke dalam suplemen secara haram bagi menurunkan berat badan dan menyembunyikan agen doping yang lain. Kehadiran bahan-bahan tersebut menyebabkan risiko kesihatan yang jelas, termasuklah ketidakseimbangan elektrolit dan ketidakfungsian ginjal, serta menyumbang kepada pelanggaran doping dalam sukan. Meskipun pemantauan pencemaran diuretik di dalam suplemen protin diperlukan secara kritikal, terdapat jurang yang ketara di dalam literatur mengenai kaedah-kaedah pengekstrakan yang optimum untuk sebatian ini. Kajian ini bertujuan untuk menilai keberkesanan bagi pelbagai jenis pelarut dalam mengekstrak sebatian diuretik daripada suplemen protin. Dengan menilai kadar pemulihan pengekstrakan dan mengoptimum pemilihan pengekstrakan pelarut, kajian ini bertujuan untuk meningkatkan kepekaan, kekhususan, dan ketepatan bagi analisis kromatografi cecair-spektrometri jisim (LC-MS/MS). Kajian ini menyiasat keberkesanan pengekstrakan dan kesan matrik bagi metanol dan acetonitril untuk mengesan diuretik di dalam protin whey menggunakan pengekstrakan cecair-cecair (LLE) berpasangan dengan LC-MS/MS. Komposisi pelarut yang berbeza anataranya 100%, 70%, dan 50% kepekatan metanol (100M, 70M, 50M) dan acetonitril (100A, 70A, 50A) telah dinilai untuk mengoptimum kadar pemulihan dan meminimumkan gangguan matrik. Hasil kajian menunjukkan bahawa metanol menunjukkan prestasi tinggi secara konsisten berbanding acetonitril dalam pengekstrakan sebatian diuretik daripada protin whey. Purata kadar pemulihan pada 100M menunjukkan yang tertinggi (49.639% to 99.735%) dengan kesan penambahan isyarat yang sederhana dan pengurangan isyarat yang minimum, ini menunjukkan keberkesanan dalam pengurangan gangguan matrik. Sama seperti 100M, 70M juga mengekalkan kesan matrik yang seimbang dan pemulihan yang boleh dipercayai (46.976% to 94.492%), menjadikan ia sebagai alternatif yang teguh bagi analisis diuretik. Manakala acetoniril pula menunjukkan kebolehubahan yang lebih besar dalam kesan matrik dan kadar pemulihan yang lebih rendah. Sebagai contoh, 100A menunjukkan pengurangan isyarat yang ketara (0.070% hingga 9.267%), ini menunjukkkan batasan dalam melarutkan diuretik daripada protin whey. Walaupun 70A memberikan profil yang lebih stabil, ia masih menunjukkan kebolehubahan (kesan matrik daripada -44.539% kepada 29.493%) berbanding metanol. Kajian ini menyerlahkan peranan penting pemilihan pelarut dalam meminimumkan kesan matrik dan memastikan kuantifikasi diuretik yang tepat dalam matriks makanan yang kompleks. Kuasa pelarut metanol yang unggul dan kepolarannya menyumbang kepada keberkesanannya dalam mengurangkan gangguan matriks berbanding dengan asetonitril. Kajian ini memberikan pandangan yang berharga untuk ahli kimia analisis dan saintis makanan yang bertujuan untuk meningkatkan ketepatan dan konsistensi analisis diuretik dalam matrik makanan, dengan itu memastikan keselamatan pengguna dan pematuhan peraturan.

 

Kata kunci: diuretik, pengekstrakan cecair-cecair, kromatografi cecair, protin whey

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