Herbalife Manufacturing, LLC, Quality Control, 20481 Crescent Bay Drive, Lake Forest, CA 92630. 2. Herbalife Nutrition, 950 West 190th Street, Torrance, CA ...
Rapid Near Infrared (NIR) Quantitation of Sodium Benzoate and Potassium Sorbate Preservatives in Aloe Concentrate 1
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Isaac Lee , Quanyin Gao , Piyush Purohit , Veronica Zarraga , Peter Chang , Gary Swanson , Keith Freel 1
Herbalife Manufacturing, LLC, Quality Control, 20481 Crescent Bay Drive, Lake Forest, CA 92630 2 Herbalife Nutrition, 950 West 190th Street, Torrance, CA 90502 3 Metrohm USA, Inc., 6555 Pelican Creek Circle, Riverview, FL 33578
Abstract
Materials and Methods
Results
In this study, a rapid NIR spectroscopy method was developed to quantify the preservatives (Sodium Benzoate and Potassium Sorbate) in flavored and unflavored aloe drinks at 0.1% level. The NIR technique has many advantages over the HPLC technique typically used in the industry. The NIR technique is time efficient with no sample preparation required and shorter sample analyzing time (< 1min) compared to the HPLC method (>120 min). This method can be applied as process analytical technology (PAT) for in situ preservative level monitoring for the production line. NIR Spectroscopy uses overtone absorptions and combination vibrations to measure the quantity of analytes. A 2 mm path length quartz cuvette was used in this study. The calibration models for each of the flavored and unflavored aloe drinks were built using the spectra of different preservative concentration standards. The calibration models were validated using twenty-eight (28) different preservative concentration samples from 0.02% to 0.16%. The results between theoretical and experimental values were comparable, indicating that the calibration model is valid. In addition, the NIR results of the flavored and unflavored aloe drink finished products were compared to that of the HPLC results. The results show the both techniques were equivalent.
The placebo of unflavored and mango flavored aloe drinks were prepared by mixing ingredients of each formula, except the preservatives. Herbalife together with Metrohm established calibration models by using six (6) different preservative concentrated standards in placebo matrix (from 0.0% to 0.2%), and we validated the calibration models with five (5) different preservative concentration standards in placebo matrix (from 0.0% to 0.15%). These five (5) standards were analyzed in triplicate to determine the precision. After validating the model, we tested the model with duplicates of six (6) preservative concentration samples (0.00%, 0.08%, 0.09%, 0.10%, 0.11%, and 0.12%). Figure 1 shows the region where the difference observed based on the preservative concentrations after applying the 2nd derivative math treatment.
Linearity of the calibration models was analyzed. Example calibration curves are shown below.
The specificity of the models was successfully evaluated by observing no preservatives detected on the blank. The precision was evaluated by analyzing five (5) different validation standards in triplicate, and RSD of the standards were below 10%. The Standard Error of Calibration (SEC) and the Standard Error of Prediction (SEP) results for the calibration and validation of models are listed in table 1. Table 1: Errors of calibration and validation of the model
Introduction Many nutritional and food products contain preservatives to maintain it fresh, and traditionally HPLC has been used to detect the preservatives. The HPLC method requires sample preparation, reagents, and longer analysis time. To enhance the lab efficiency, NIRS method has been developed to quantify the amount of two (2) preservatives (Sodium Benzoate and Potassium Sorbate). The analysis time of NIR method is much shorter than the HPLC method. Furthermore, the technique is environmentally friendly, which does not require reagents or harsh chemicals. By using the NIR method, we will save the cost of reagents, time, and hazardous waste disposal. FT-IR, which uses mid-IR region, can give fundamental information such as functional groups identification and structural characterization. However, it cannot quantitate such low amount of analytes in a sample. NIR uses higher energy states of vibration by combining fundamental vibration and overtones. It finds wavelength regions that show differences depending on the concentration of the samples and applies appropriate math treatments, such as first and second derivatives, to correlate with the change of preservative concentration.
Conclusion The calibration and validation of each model shows the method is accurate with low errors (SEC and SEP). Also, the validation results generated in our lab demonstrates that the test method is specific, linear, accurate, and precise. The results between the NIR and HPLC instruments are comparable. All results show the NIRS models have been successfully established and validated. The NIR instrument gives accurate and precise results more rapidly than HPLC.
Unflavored Aloe Drink Mango flavored Aloe Drink
Figure 1: Comparison of spectra of different preservative concentration samples after applying the 2nd derivative math treatment (red-0.00%, blue-0.08%, teal-0.09%, magenta-0.10%, green-0.11%, and purple-0.12)%
These samples were prepared by mixing different amount of preservatives in the placebos (Unflavored and Mango Flavored Aloe Drink). One of the samples was a blank, which did not contain any preservatives. The specificity of the model was evaluated using the blank. The first set of eight (8) samples only contained different concentrations of Sodium Benzoate from 0.02% to 0.16%. The second set of eight (8) samples only contained different concentrations of Potassium Sorbate from 0.02% to 0.16%. The third set of eleven (11) samples contained different mixtures of both preservatives from 0.08% to 0.14%. Also, four (4) different aloe finished products from each formula were analyzed using both instruments (NIR and HPLC) for comparison.
Sodium Benzoate Potassium Sorbate Sodium Benzoate Potassium Sorbate
SEP
0.00% 0.01% 0.00% 0.00%
0.00% 0.01% 0.00% 0.00%
The model was additionally validated by our lab with twenty-eight (28) prepared samples. The theoretical values and the actual test results were compared, and the range of bias are listed in table 2. Table 2: Range of bias on validation with twenty eight (28) samples Unflavored Aloe Drink
Additional validation for each of the model was performed with twenty-eight (28) different preservative concentration samples. Validating the models with more samples would give assurance on validity of the model and method ruggedness.
% % % %
SEC
Mango Flavored Aloe Drink
% % % %
Sodium Benzoate Potassium Sorbate Sodium Benzoate Potassium Sorbate
Highest
Lowest
0.003 0.007 0.006 0.004
0.000 0.000 0.000 0.000
The four (4) samples of the each formula were analyzed using two (2) instruments (NIR vs. HPLC) for comparison. Results are shown in table 3. Table 3: Comparison of Preservative results between HPLC and NIR Sample Name Unflavored Aloe Unflavored Aloe Unflavored Aloe Unflavored Aloe Mango Flavored Mango Flavored Mango Flavored Mango Flavored
Sample# 1 Sample# 2 Sample# 3 Sample# 4 Aloe Sample# Aloe Sample# Aloe Sample# Aloe Sample#
1 2 3 4
% Potassium Sorbate (HPLC)
% Potassium Sorbate (NIR)
% Sodium Benzoate (HPLC)
% Sodium Benzoate (NIR)
0.10 0.10 0.10 0.10 0.09 0.08 0.08 0.08
0.09 0.10 0.09 0.10 0.10 0.10 0.10 0.10
0.11 0.11 0.11 0.11 0.09 0.09 0.09 0.09
0.10 0.11 0.12 0.09 0.10 0.10 0.10 0.09
