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 UPLC-DAD Method for Quantitative Analysis of Coumarin, trans-Cinnamic Acid, trans-Cinnamaldehyde, and Eugenol in Encapsulated Cinnamon Flavoring Powder.

BACKGROUND: Cinnamon is a popular spice used in food products. Its flavor varies by its chemical profile. Cinnamon flavoring powder is a unique form of material with essential oil encapsulated in wall material, which improves the stability and homogeneity but also increases the difficulties for analysis. A specific and rapid method is needed to analyze the main components for its quality and safety. OBJECTIVE: An analytical method for the quantification of cinnamon flavoring powder was developed and validated. The characteristic components for analysis were selected as coumarin, trans-cinnamic acid, trans-cinnamaldehyde, and eugenol. METHODS: This quantitation method with ultra-performance liquid chromatography coupled with diode array detector analysis was achieved by material extraction followed by chromatographic separation on C18 columns eluted with a gradient acetonitrile-water mobile phase. The detected wavelength was determined as 280 nm. RESULTS: Linear regression of calibration curves for each component was validated (R2 > 0.9995). The specificity, LOD and LOQ, precision, accuracy, and ruggedness of the developed method were also evaluated. CONCLUSIONS: Such an approach is applicable for the simultaneous determination of these four characteristic constituents in cinnamon flavoring powder used in manufacturing and quality control of nutritional products. HIGHLIGHTS: This study describes the selection of four components for analysis, the efficient extraction of them from cinnamon flavoring powder, and the rapid quantitation of these four characteristic components in these materials.

Field Identification of Matricaria chamomilla using a Portable qPCR System.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

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.

Development of a Differential Multiplex PCR Assay for the Supplemental Identification of Different Sources of Proteins.

BACKGROUND: Differentiation of proteins from multiple sources provides challenges in the accuracy using multiple and often disputed protein identification methods. The U.S. Pharmacopeia Food Chemical Codex does not include monographs for many protein sources, including milk proteins and soy protein isolate. Monographs that are included for proteins do not list a single comprehensive identification method but instead rely on a combined assessment of ash (total), fat, lactose, loss on drying, and protein content. A fast, inexpensive, and accurate protein source assay is tantamount to prevention of economic adulteration in protein powders. OBJECTIVE: This study describes the development of a novel method to identify and differentiate animal proteins (cow protein powders as milk protein and whey protein) and plant proteins (soy protein powders). These proteins powders are of high importance to the food and dietary supplement industries, as they encompass the highest grossing and fastest growing protein sources in the global protein powder market. METHODS: The developed method uses PCR amplification and gel electrophoresis of short chain DNA fragments found in processed protein powders to identify and differentiate the source of each powder. The original development was performed using reference materials of known identity and tested against an inclusivity panel of protein powders from commercial sources. Bands were identified using the Agilent Tapestation 4200 and Tapestation Analysis Software A.02.02 (SR1) using proprietary band analysis. RESULTS: The developed method was found to be specific for the identification of each protein source, passing a computational (National Center for Biotechnology Information Basic Local Alignment Search Tool) exclusivity panel and an experimental inclusivity panel. The method was also able to detect multiple adulterants in concentrations as low as 1% (w/w). CONCLUSIONS: The developed method is fast, inexpensive, and accurate (100%) for the supplemental identification of cow and soy proteins and able to detect adulteration as low as 1% (w/w). HIGHLIGHTS: A new method can identify cow and soy proteins, and detect low levels of adulteration using DM-PCR.

Validation of a Targeted PCR Method for Raw and Processed Botanical Material Identification: An Example Using Matricaria chamomilla (Chamomile).

Background: A requirement of current good manufacturing practices for dietary supplements is that manufacturers must identify their dietary ingredients. DNA-based methods can provide species-level authentication that may sometimes be difficult to achieve using conventional morphological and chemical analysis methods. However, because of varying levels of DNA degradation in botanical materials, many commercial tests fail to generate consistent test results across all types of botanical materials. AOAC published guidelines for validation of botanical identification methods and proposed probability of identification (POI) as a method performance parameter. However, few DNA-based botanical authentication methods in the literature follow these guidelines and evaluate POI. Objective: To provide a targeted PCR method validation example that follows AOAC guidelines for validation of botanical identification methods. Methods: Using Matricaria chamomilla (chamomile) as an example, we performed a single-laboratory validation for a targeted PCR method that aimed to identify both raw and processed chamomile materials. The performance parameters of the test were evaluated by carrying out an inclusivity/exclusivity study and a Specified Superior Test Material/Specified Inferior Test Material study to demonstrate that the method's POI meets industry requirements. Results: The chamomile samples were identified by the method and achieved a POI greater than 0.9 with respect to all types of chamomile botanical materials. Conclusions: The method was validated for DNA-based identification of raw and processed chamomile materials, such as sterilized powders and extracts. Highlights: This work will provide insight for laboratories and manufacturers that aim to develop and validate DNA-based botanical identification methods.

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.

Quantitative Analysis of Aloins and Aloin-Emodin in Aloe Vera Raw Materials and Finished Products Using High-Performance Liquid Chromatography: Single-Laboratory Validation, First Action 2016.09.

A single-laboratory validation study is described for a method of quantitative analysis of aloins (aloins A and B) and aloe-emodin in aloe vera raw materials and finished products. This method used HPLC coupled with UV detection at 380 nm for the aloins and 430 nm for aloe-emodin. The advantage of this test method is that the target analytes are concentrated from the sample matrix (either liquid or solid form) using stepwise liquid-liquid extraction (water-ethyl acetate-methanol), followed by solvent evaporation and reconstitution. This sample preparation process is suitable for different forms of products. The concentrating step for aloins and aloe-emodin has enhanced the method quantitation level to 20 parts per billion (ppb). Reversed-phase chromatography using a 250 × 4.6 mm column under gradient elution conditions was used. Mobile phase A is 0.1% acetic acid in water and mobile phase B is 0.1% acetic acid in acetonitrile. The HPLC run starts with a 20% mobile phase B that reaches 35% at 13 min. From 13 to 30 min, mobile phase B is increased from 35 to 100%. From 30 to 40 min, mobile phase B is changed from 100% back to the initial condition of 20% for re-equilibration. The flow rate is 1 mL/min, with a 100 µL injection volume. Baseline separation (Rs > 2.0) for aloins A and B and aloe-emodin was observed under this chromatographic condition. This test method was validated with raw materials of aloe vera 5× (liquid) and aloe vera 200× (powder) and finished products of aloe concentrate (liquid) and aloe (powder). The linearity of the method was studied from 10 to 500 ppb for aloins A and B and aloe-emodin, with correlation coefficients of 0.999964, 0.999957, and 0.999980, respectively. The test method was proven to be specific, precise, accurate, rugged, and suitable for the intended quantitative analysis of aloins and aloe-emodin in raw materials and finished products. The S/N for aloins A and B and aloe-emodin at 10 ppb level were 12, 10, and 8, respectively, indicating our conservative LOD level at 10 ppb (the typical LOD level S/N is about 3). The S/N for aloins A and B and aloe-emodin at the 20 ppb level were 17, 14, and 16, respectively, indicating our conservative LOQ level at 20 ppb (the typical LOQ level S/N is about 10). The stock standard solution of a mixture of aloins and aloe-emodin and a working standard solution were found to be stable for at least 19 days when stored refrigerated at 2-8°C, with a recovery of 100 ± 5%.