Research Article
A Simplified, Specific HPLC Method of Assaying Thiamine and Riboflavin in Mushrooms
Table 1
Method validation results.
| | Validation Parameters | Riboflavin (B2) | Thiamine (B1) |
| | Detection Wavelength | 268 nm | 245 nm | | Retention Time | ~ 6.0-7.0 min | ~ 9.0 -9.5 min | | Peak Purity-Standard | PA: 0.166 < PT: 0.308 | PA: 0.166 < PT: 0.285 | | Peak Purity-Spiked | PA: 0.300 < PT: 0.316 | PA: 0.225 < PT: 0.307 | | Linearity | y = 523393x-3627, R2 = 1.00 | y = 288713x-2270, R2 = 1.00 | | Limit of detection (LOD) | 0.029μg/mL | 0.043μg/mL | | Limit of quantitation (LOQ) | 0.088μg/mL | 0.132μg/mL | | %Recovery & Precision (Mean ±SD) | 104.88±2.04 (103% -107%) | 104.45±4.5 (101% – 109%) | | System Precision (%RSD of Area) | 0.63 | 1.17 | | %Recovery & Intermediate Precision | 103.35±2.80 (101% – 106%) | 101.97±2.43 (99% -104%) | | Solution stability (Standard & Sample) | 72 hours at controlled cold temperature/Refrigerator |
| | Robustness data | Resolution between Riboflavin and Thiamine |
| | HPLC Column 1 (Xterra MS C-18) | 4.6 (Retention Time: 7.0/9.2) | | HPLC Column 2 (X-Select C-18) | 3.5 (Retention Time: 7.0/8.5) | | Mobile Phase Ratio (78:22) | 4.3 (Retention Time: 6.9/9.0) | | Mobile Phase Ratio (80:20) | 4.6 (Retention Time: 7.0/9.2) | | Mobile Phase Ratio (82:18) | 4.6 (Retention Time: 8.2/9.9) |
| | Sample Analysis (Mean in μg/g ±SD) (Shitaki mushrooms) | Day 1: 5.95±0.44 | Day 1: 0.93±0.07 | | Day 2: 6.03±0.27 | Day 2: 1.17±0.001 |
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PA: purity angle and PT: purity threshold, blend (six-month-old) of six natural strains of Shitaki mushrooms.
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