https://doi.org/10.3390/molecules28020765
”
2.1. Validation of Quantitative HPLC Analysis
To allow head-to-head comparison of the qNMR analysis of carbohydrates (Figure 1) with more conventional analytical techniques, HPLC (PMP-HPLC-UV, HPLC-RID and HPLC-ELSD) assays were established. As shown in Figure 2, which are typical chromatograms, glucose and maltose were well separated by PMP-HPLC, and all four analytes were well resolved by HPLC-RID and HPLC-ELSD. For the PMP-HPLC-UV method, the linearity range of glucose and maltose was 0.01–1.00 mg/mL with correlation coefficients higher than 0.9998 (Table 1). Intra-day variations were less than 0.7%, and inter-day variations did not exceed 1.2%. The repeatability was less than 2.3%, and the stability was less than 1.9%. Sample recovery was less than 3.3% (Table 2). By HPLC-RID analysis, the linearity ranges for fructose, glucose, sucrose and maltose were 0.10–2.08, 0.10–2.13, 0.10–2.07 and 0.10–2.05 mg/mL, respectively (Table 1). Within these ranges, the calibration function correlation coefficients were better than 0.9993 for all analytes. Intra-day variations were less than 1.3%, and inter-day variations were no more than 2.2%. The repeatability was less than 2.2%, and the stability was less than 1.5%. Sample recovery was less than 3.2% (Table 2). Meanwhile, the linearity range of fructose was 0.10–2.00 mg/mL by the HPLC-ELSD method, while the linearity range of glucose, sucrose and maltose was 0.20–4.00 mg/mL (Table 1). Calibration correlation coefficients of all analytes were better than 0.9994. Intra-day variations were less than 1.7%, and inter-day variations did not exceed 2.6%. The results of repeatability, stability and recovery were listed in Table 2, which were all acceptable and reliable.

Figure 1. Structures of TSP (internal standard, IS) and sugars. The analyte fructose, glucose, sucrose and maltose were numbered 1–4, respectively. qNMR resonances of highlighted protons were used for qNMR-based analyte quantitation.

Figure 2. Chromatograms of standard solution (A) and sample solutions (B) of sugars from YQFM for PMP-HPLC (a), HPLC-RID (b) and HPLC-ELSD (c). Numbers 1–4 correspond to fructose, glucose, sucrose and maltose, respectively.
Table 1. HPLC-based calibration function parameters for compounds, including regression equations, correlation coefficients (r) and linearity range.
Method | Compound | Y (Peak Area) = kcA + d | r | Linearity Range (mg/mL) | LLOQ (mg/mL) |
---|---|---|---|---|---|
PMP-HPLC | Glucose | Y = 17,739X + 6829.8 | 0.9998 | 0.01~1.00 | 0.01 |
Maltose | Y = 9116.4X + 1027.0 | 1.0000 | 0.01~1.00 | 0.01 | |
HPLC-RID | Fructose | Y = 103,246X + 6774.8 | 0.9998 | 0.10~2.08 | 0.10 |
Glucose | Y = 116,724X + 2647.4 | 0.9993 | 0.10~2.13 | 0.10 | |
Sucrose | Y = 83,425.0X + 8358.1 | 0.9994 | 0.10~2.07 | 0.10 | |
Maltose | Y = 67,678.0X−2551.7 | 0.9997 | 0.10~2.05 | 0.10 | |
HPLC-ELSD | Fructose | Y = 1.3246X +0.6774 | 0.9999 | 0.10~2.00 | 0.10 |
Glucose | Y = 1.3425X + 0.8358 | 0.9994 | 0.20~4.00 | 0.20 | |
Sucrose | Y = 1.0548X−0.6375 | 0.9995 | 0.20~4.00 | 0.20 | |
Maltose | Y = 1.1267X−0.2551 | 0.9996 | 0.20~4.00 | 0.20 |
Table 2. HPLC assay accuracy for compounds. Inter-day recovery values (n = 6) expressed in percentage amount added (mean ± RSD).
Method | Compound | Recovery (%±RSD) |
---|---|---|
PMP-HPLC | glucose | 99.4 ± 3.3 |
maltose | 102.4 ± 2.9 | |
HPLC-RID | fructose | 100.1 ± 0.4 |
glucose | 101.4 ± 3.2 | |
sucrose | 97.3 ± 2.5 | |
maltose | 99.7 ± 2.8 | |
HPLC-ELSD | fructose | 98.6 ± 1.2 |
glucose | 100.6 ± 1.5 | |
sucrose | 99.6 ± 3.0 | |
maltose | 100.1 ± 2.5 |
“