Single-Laboratory Validation Study of a Proton NMR Method for the Determination of L-Arginine, L-Citrulline, and Taurine Contents in Dietary Supplements.

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.

Development and validation of a probe-based qPCR method to prevent parsley leaf material misidentification.

Parsley (Petroselinum crispum) leaf is an herb widely consumed for its health benefits. Due to similar morphological and chemical profiles, celery leaf may be a source of substitution in commercial parsley materials. In order to detect this substitution, the present work characterized parsley and celery leaf at the cultivar level by physical, chemical and DNA approaches. In contrast to the variations observed in physical appearances and chemical profiles that make verification of authenticity difficult, consistent differences observed between their DNA sequences are suitable for verifying parsley material authenticity. To identify parsley and detect celery simultaneously, a multiplex qPCR assay was developed and validated with respect to efficiency and specificity. Further testing indicated the assay can be used to detect 1% (w/w) celery in parsley materials with a probability of detection greater than 0.9. The developed method is well-suited for routine quality control to prevent parsley material misidentification in commercial trade.

Single-Laboratory Validation of a Two-Tiered DNA Barcoding Method for Raw Botanical Identification.

Background: The applications of deoxyribonucleic acid (DNA) barcoding methods have been extended from authenticating taxonomic provenance of animal products to identifying botanicals used as herbal medicine and in botanical dietary supplements. DNA barcoding methods for botanical identification must be adequately validated to meet regulatory compliance. Objective: The goal of this study is to provide a validation protocol for a two-tiered DNA barcoding method that aims to identify raw botanicals. Methods: A barcode database was computationally validated to define the barcode combinations that can unambiguously identify botanicals in the database. A maximum variation sampling technique was used to capture a wide range of perspectives relating to DNA barcode-based botanical identification, including plant parts and species distance, for the experimental validation. Twenty-two authenticated botanicals were purposively sampled from different plant parts-covering both closely related and distantly related species-to validate the two-tiered DNA barcoding method. The performance of the method was assessed on accuracy, precision, ruggedness, and uncertainty. Results: High accuracy (100%) and precision (1.0) were obtained from the validation samples. The method was also found to be rugged and have acceptable uncertainty. Conclusions: The method was validated and suitable for DNA-based identification of botanical raw materials listed in the current database. Highlights: This work will provide support guidance for manufacturers and regulatory policy makers to implement equivalent validated and compliant DNA-based testing in quality control processes to improve botanical raw material identification and authentication.

Single-Laboratory Validation of a Method for Determination of Ergocalciferol in Protein Drink Powders and Tablets by LC-MS/MS.

Background: Currently, there is a lack of validation studies available in the literature for the determination of ergocalciferol, especially for those using a direct extraction technique. The current official methodologies for the quantification of ergocalciferol require saponification, liquid-liquid extraction, or both, thus requiring experienced technicians and specialized reflux equipment. This work provides a method that is more easily accessible to laboratories without these resources while still achieving the robustness needed for a successful validation of low levels of ergocalciferol in complex matrixes. Objective: A single-laboratory validation study was conducted for a rapid quantification method of ergocalciferol in protein drink powders and tablets. Methods: The method uses an LC-MS/MS with multimode source utilizing atmospheric pressure chemical ionization positive ionization mode. For both protein drink powders and tablets, the procedure consisted of a liquid extraction step using dimethyl sulfoxide and methanol. Isotopically labeled ergocalciferol was used as an internal standard to correct for signal depression caused by matrix interference. Results: This LC-MS/MS method was found to be accurate, precise, linear (from 0.01 to 0.3 µg/mL), rugged, and suitable for protein drink powders and tablets. Conclusions: The method was validated and is suitable for accurate quantification of ergocalciferol in tablet and protein powder products. Highlights: This work provides a validated method for accurate quantification of ergocalciferol in complex matrixes using a direct extraction technique. This may benefit quality control laboratories in the food and nutraceutical industries, where simple and efficient methodology is key to optimal functioning.

Visualization of DNA in highly processed botanical materials.

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is "invisible" by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the "invisible" DNA in botanical extracts. The size of the "invisible" DNA fragments in botanical extracts was around 20-220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future.