单实验室验证研究质子核磁共振法测定膳食补充剂中 L-精氨酸、L-瓜氨酸和牛磺酸含量。
Single-Laboratory Validation Study of a Proton NMR Method for the Determination of L-Arginine, L-Citrulline, and Taurine Contents in Dietary Supplements.
机构信息
Herbalife Manufacturing, LLC, 20481 Crescent Bay Drive, Lake Forest, CA 92630, USA.
Herbalife International of America, 950 West 190th Street, Torrance, CA 90502, USA.
出版信息
J AOAC Int. 2020 Jul 1;103(4):1140-1147. doi: 10.1093/jaoacint/qsaa002.
BACKGROUND
A quantitative NMR (qNMR) method can provide rapid analysis compared to chromatographic methods. Sample preparation steps are relatively simpler and run time is shorter. Rapid analysis methods for release tests in quality control laboratories are very important for laboratory efficiency. Here, we describe a single-laboratory validation study for a rapid qNMR analysis of L-arginine, L-citrulline, and taurine in powdered and tablet dietary supplement products.
OBJECTIVES
This validation work is to provide documented evidence for the qNMR method validity as well as method performance.
METHODS
The method used Bruker 400 MHz high-resolution proton NMR spectroscopy for simultaneous determination of L-arginine, L-citrulline, and taurine contents in dietary supplement product 1 (powder) and dietary supplement product 2 (tablet). The absolute NMR quantitation is based on a principle of universal proton response intensity correlation with the number of protons in each target analyte (amino acids) vs. that of a reference standard (maleic acid).
RESULTS
The test method performance was validated with dietary supplement-1 (powder) and dietary supplement-2 (tablet). The linearity of the method was studied from about 360 mg/g to about 675 mg/g of L-arginine; from about 15 mg/g to about 30 mg/g of L-citrulline; and from about 20 mg/g to about 40 mg/g of taurine in dietary supplement-1, and from about 15 mg/g to about 30 mg/g of taurine in dietary supplement-2. The coefficients of determination (R2) are 1.0000 for L-arginine, 0.9967 for L-citrulline, and 0.9995 for taurine in dietary supplement-1 and 0.9903 for taurine in dietary supplement-2. The accuracies measured from the sample matrices are 102%, 101%, and 100% average recoveries for 80%, 100%, and 120% concentration levels of L-arginine, 105%, 105%, and 103% average recoveries for 80%, 100%, and 120% concentration level of L-citrulline, and 101%, 102%, and 100% average recoveries of taurine for 80%, 100%, 120% concentration levels in dietary supplement-1; and 95, 98%, and 93% average recoveries of taurine for 80%, 100%, 120% concentration levels in dietary supplement-2, respectively. The precisions (RSD) are 1% for L-arginine, 5% for L-citrulline, and 2% for taurine in dietary supplement -1, respectively; and 4% for taurine in dietary supplement-2. The ruggedness of the test method is within 2%, 4%, and 2% for L-arginine, L-citrulline, and taurine for dietary supplement -1, respectively, and within 4% for dietary supplement-2. The method is specific for the quantitation of each nutrient with no background interference from the matrix for the proton peaks of L-arginine, L-citrulline, taurine, and maleic acid (standard).
CONCLUSIONS
The test method is proven to be specific, precise, accurate, rugged, and suitable for intended quantitative analysis of L-arginine, L-citrulline, and taurine in powdered and tablet finished products.
HIGHLIGHTS
The simultaneous determination of all three nutrients of L-arginine, L-citrulline, and taurine using proton NMR provides rapid analysis for quality control release tests that is more efficient versus that of two HPLC methods. Previously, our laboratory was using one HPLC method to analyze L-arginine and L-citrulline while using a second HPLC method to analyze taurine. That approach required two HPLC instruments and two analysts for parallel analysis that takes 2 days using volatile and flammable solvents for extraction and chemical derivatization. This rapid NMR method can analyze the sample "as is" with results obtained in less than 4 h, and is efficient, safe, and environmentally friendly. The initial higher NMR instrument investment versus two HPLC instruments is rewarded with high returns for continued quality control tests.
背景
与色谱方法相比，定量 NMR（qNMR）方法可以提供快速分析。样品制备步骤相对更简单，运行时间更短。对于质量控制实验室的放行测试，快速分析方法对于实验室效率非常重要。本文描述了一种用于粉末状和片剂膳食补充剂产品中 L-精氨酸、L-瓜氨酸和牛磺酸的快速 qNMR 分析的单实验室验证研究。
目的
这项验证工作旨在为 qNMR 方法的有效性以及方法性能提供文件证据。
方法
该方法使用 Bruker 400 MHz 高分辨率质子 NMR 光谱法同时测定膳食补充剂产品 1（粉末）和膳食补充剂产品 2（片剂）中 L-精氨酸、L-瓜氨酸和牛磺酸的含量。绝对 NMR 定量基于普遍质子响应强度与每个目标分析物（氨基酸）中质子数量与参考标准（马来酸）中质子数量的相关性原理。
结果
使用膳食补充剂 1（粉末）和膳食补充剂 2（片剂）对测试方法的性能进行了验证。方法的线性从大约 360 mg/g 到大约 675 mg/g 的 L-精氨酸；从大约 15 mg/g 到大约 30 mg/g 的 L-瓜氨酸；从大约 20 mg/g 到大约 40 mg/g 的牛磺酸在膳食补充剂 1 中，以及从大约 15 mg/g 到大约 30 mg/g 的牛磺酸在膳食补充剂 2 中进行了研究。L-精氨酸、L-瓜氨酸和牛磺酸在膳食补充剂 1 中的决定系数（R2）分别为 1.0000、0.9967 和 0.9995，在膳食补充剂 2 中的决定系数为 0.9903。从样品基质中测量的准确度为 80%、100%和 120%浓度水平的 L-精氨酸的 102%、101%和 100%平均回收率，80%、100%和 120%浓度水平的 L-瓜氨酸的 105%、105%和 103%平均回收率，80%、100%和 120%浓度水平的牛磺酸的 101%、102%和 100%平均回收率在膳食补充剂 1 中；80%、100%和 120%浓度水平的牛磺酸的 95%、98%和 93%平均回收率在膳食补充剂 2 中，分别。精密度（RSD）分别为膳食补充剂 1 中 L-精氨酸的 1%、L-瓜氨酸的 5%和牛磺酸的 2%，以及膳食补充剂 2 中牛磺酸的 4%。该方法的耐用性在膳食补充剂 1 中分别为 L-精氨酸、L-瓜氨酸和牛磺酸的 2%、4%和 2%，在膳食补充剂 2 中为 4%。该方法对每个营养素的定量具有特异性，不存在基质对 L-精氨酸、L-瓜氨酸、牛磺酸和马来酸（标准）质子峰的背景干扰。
结论
该测试方法已被证明具有特异性、精确性、准确性、耐用性，适用于粉末状和片剂成品中 L-精氨酸、L-瓜氨酸和牛磺酸的定量分析。
重点
使用质子 NMR 同时测定所有三种营养素 L-精氨酸、L-瓜氨酸和牛磺酸，为质量控制放行测试提供了比两种 HPLC 方法更高效的快速分析。此前，我们实验室使用一种 HPLC 方法来分析 L-精氨酸和 L-瓜氨酸，而使用另一种 HPLC 方法来分析牛磺酸。该方法需要两种 HPLC 仪器和两名分析师同时进行平行分析，使用挥发性和易燃溶剂进行提取和化学衍生化，需要两天时间。这种快速 NMR 方法可以“原样”分析样品，结果在不到 4 小时内获得，并且高效、安全、环保。与两台 HPLC 仪器相比，初始较高的 NMR 仪器投资将获得持续的质量控制测试的高回报。
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