# High-Performance Liquid Chromatography–Diode Array Detection Combined with Chemometrics for Simultaneous Quantitative Analysis of Five Active Constituents in a Chinese Medicine Formula Wen-Qing-Yin

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Chemicals and Reagents

^{−1}. All stock solutions were kept at 4 °C in the refrigerator until they were utilized.

#### 2.2. Chromatographic Instrument and Conditions

^{TM}C18 column (5 μm, 200 mm × 4.6 mm) with the column temperature of 30 °C and the column pressure at 6.7 MPa. The flow rate was set at 1.00 mL min

^{−}

^{1}and the injection volume was 20 μL. In order to enhance the resolution and improve the peak symmetry, small amounts of formic acid were added to the aqueous phase with the optimal concentration of 1.0 mL L

^{−1}. The mobile phase was composed of 0.1% formic acid aqueous solution (solvent A) and acetonitrile solution (solvent B). Under the isocratic elution condition of 30% B, all analytes were eluted within 10 min in isometric elution mode.

#### 2.3. Sample Preparation Procedures

_{8}* (8

^{5}) to minimize the potential co-linear factor. Three spiked prediction samples were prepared by transferring the processed WQY actual sample (manufactured by Sun Ten Pharmaceutical Co., Ltd., Taipei, China) and the appropriate volume of the working solution to a 10 mL volumetric flask and diluting it with methanol. Considering that the concentrations of BAI and BER in the WQY real sample are much higher than the concentrations of HMF, PAE, and FER, the real samples of WQY were added to the predicted samples in two concentrations, diluted 20 times to detect HMF, PAE, and FER (P01–P03), and 50 times to detect BAI and BER (P04-P06). The concentrations of the calibration set samples are shown in Table S2.

#### 2.4. Theory

#### 2.4.1. Trilinear Component Model

**X**(I × J × K). The array

**X**can be represented by a mathematical equation as follows:

_{ijk}is the element of the three-way array

**X**(I × J × K), and it represents the response value of the sample k at elution time i and spectral channel J. a

_{i}

_{n}, b

_{jn}, c

_{k}

_{n}, and e

_{ijk}are the corresponding elements of normalized chromatographic matrix

**A**(I × N), normalized UV spectral matrix

**B**(J × N), and relative concentration matrix C (K×N) elements and three-way residue array

**E**(I × J × K).

#### 2.4.2. ATLD Algorithm

**A**and

**B**) and the relative concentration matrix (

**C**) by using the Moore–Penrose generalized inverse based on singular value decomposition:

#### 2.4.3. ATLD-MCR Algorithm

_{ini}. Then, based on the MCR strategy, the retention time profiles matrix A

_{k}of each sample is obtained through the optimized least square calculation (see Equation (5)). X

_{..k}is the slice matrix of each sample. Finally, qualitative information (A

_{k}and B) and quantitative information (C) are obtained by the following equation:

## 3. Results and Discussion

#### 3.1. General Considerations of the HPLC-DAD Experiment

#### 3.2. Quantification of Five Active Constituents in WQY

#### 3.3. Accuracy and Precision

**C**) to the real concentrations of the analytes in calibration samples, the pseudo-univariate calibration curves can be built, and then the absolute concentration of the component of interest in the unknown samples can be obtained by the established linear regression equations. The linear range and regression equation of the target analytes are shown in Table S4 and the prediction results of five target analytes based on ATLD and ATLD-MCR algorithms are summarized in Table 1; also, the detailed spiked concentration and recoveries of the six prediction samples are shown in Table S5.

^{−1}. For ATLD-MCR algorithm, the r values are between 0.9973 and 0.9995 with the RMSEPs less than 3.04 μg mL

^{−1}; the average recoveries ranged from 88.6 to 101.6% with the SDs <10.3%. The results obtained are relatively close. The RMSEPs values of ATLD-MCR are small, and all the recoveries are within acceptable ranges, indicating that the predicted concentrations are consistent with the standard concentrations, which are generally satisfactory.

#### 3.4. Repeatability and Reproducibility

#### 3.5. Analytical Figures of Merit

^{4}and 1.24 × 10

^{5}mAu mL μg

^{−1}for ATLD and 7.74 × 10

^{3}and 7.93 × 10

^{4}mAu mL μg

^{−1}for ATLD-MCR, and the SEL values of each analyte obtained by ATLD and ATLD-MCR are in the range of 0.20–0.56 and 0.13–0.35. It is worth noting that the lower SEL values of the target analytes are due to the severe impact of peak overlaps and unknown interferences. The LOD ranges of ATLD and ATLD-MCR are 0.07–11.67 µg mL

^{−1}and 0.22–4.53 µg mL

^{−1}, respectively. As can be seen, the results of LOD values obtained by ATLD are acceptable, except that the LOD of BER is lower than the minimum concentration of the calibration samples, which is due to the influence of time shifts. At the same time, the results obtained by the ATLD-MCR algorithm are reasonable and satisfactory.

#### 3.6. Evaluation of Two Methods

#### 3.7. Analysis of the Other WQY Samples

## 4. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Conflicts of Interest

## Abbreviations

HPLC-DAD | high performance liquid chromatography-diode array detection |

ATLD | alternating trilinear decomposition |

ATLD-MCR | alternating trilinear decomposition assisted multivariate curve resolution |

WQY | Wen-Qing-Yin |

TCM | traditional Chinese medicine |

RMSEP | relative root mean square error of prediction |

SD | standard deviation |

RSD | relative standard deviation |

SEL | selectivity |

SEN | sensitivity |

LOD | limit of detection |

LOQ | limit of quantitation |

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**Figure 1.**(

**a**) Two−dimensional contour profile of a mixed standard solution (C06); (

**b**) the elution time profiles at 260 nm for eight calibration samples; (

**c**) multi−channel chromatographic profile of a mixed standard solution (C06); (

**d**) the elution time profiles at 260 nm for six predicted samples.

**Figure 2.**The normalized chromatograms (

**a1**,

**a2**), the normalized spectrograms (

**b1**,

**b2**), and the relative concentration profiles (

**c1**,

**c2**) resolved by the ATLD algorithm from two three-way subarrays.

**Figure 3.**The normalized chromatograms (

**a1**,

**a2**), the normalized spectrograms (

**b1**,

**b2**), and the relative concentration profiles (

**c1**,

**c2**) resolved by the ATLD−MCR algorithm from two three−way subarrays.

**Table 1.**Accuracy of five target analytes in spiked prediction samples (P01–06) of WQY commercial products using ATLD and ATLD-MCR methods.

HMF | PAE | FER | BAI | BER | |
---|---|---|---|---|---|

ATLD | |||||

r
^{a} | 0.9991 | 0.9993 | 0.9996 | 0.9969 | 0.9978 |

AVG ± S.D.% ^{b} | 94.7± 2.5 | 91.8 ± 5.0 | 93.9 ± 3.5 | 104.3 ± 9.7 | 112.5 ± 5.1 |

RMSEP (µg mL^{−}^{1}) ^{c} | 0.20 | 2.07 | 0.38 | 3.94 | 4.05 |

ATLD-MCR | |||||

r | 0.9992 | 0.9995 | 0.9993 | 0.9985 | 0.9973 |

AVG ± S.D.% | 93.3 ± 1.4 | 92.4 ± 5.7 | 88.6 ± 3.5 | 101.6 ± 9.9 | 96.8 ± 10.3 |

RMSEP (µg mL^{−}^{1}) | 0.30 | 1.85 | 0.69 | 3.04 | 1.53 |

^{a}r represents correlation coefficients.

^{b}AVG ± S.D. is average spiked recovery ± standard deviation.

^{c}RMSEP represents relative root mean square error of prediction. RMSEP = $\sqrt{\sum _{n=1}^{N}{\left({c}_{pre}-{c}_{act}\right)}^{2}/{N}_{p}}\times 100\%$, ${c}_{pre}$ and ${c}_{act}$ are the predicted and actual concentrations, respectively, and ${N}_{p}$ is the number of prediction samples.

**Table 2.**The figures of merit and precision for determination of five analytes in selected WQY samples using ATLD and ATLD-MCR methods.

Statistic Parameter | Analytical Compounds | ||||
---|---|---|---|---|---|

HMF | PAE | FER | BAI | BER | |

ATLD | |||||

SEL ^{a} | 0.49 | 0.52 | 0.56 | 0.43 | 0.20 |

SEN ^{b}/mL µg^{−1} | 8.46 × 10^{4} | 1.04 × 10^{4} | 1.24 × 10^{5} | 3.00 × 10^{4} | 2.46 × 10^{4} |

LOD ^{c}/µg mL^{−1} | 0.95 | 1.84 | 0.07 | 4.94 | 11.67 |

LOQ ^{d}/µg mL^{−1} | 2.85 | 5.59 | 0.22 | 14.98 | 35.37 |

Intra-day (RSD % n = 3) | 9.65 | 3.24 | 0.67 | 2.12 | 5.22 |

Inter-day (RSD % n = 3) | 12.47 | 2.72 | 4.47 | 3.16 | 40.82 |

ATLD-MCR | |||||

SEL | 0.15 | 0.23 | 0.35 | 0.13 | 0.19 |

SEN/mL µg^{−1} | 3.09 × 10^{4} | 7.74 × 10^{3} | 7.93 × 10^{4} | 9.11 × 10^{3} | 2.24 × 10^{4} |

LOD/µg mL^{−1} | 0.22 | 1.12 | 0.17 | 4.53 | 0.84 |

LOQ/µg mL^{−1} | 0.65 | 3.40 | 0.51 | 13.72 | 2.54 |

Intra-day (RSD % n = 3) | 3.04 | 1.12 | 1.33 | 1.83 | 0.36 |

Inter-day (RSD % n = 3) | 2.90 | 1.64 | 2.65 | 2.14 | 0.92 |

^{a}${\mathrm{SEL}}_{n}=\{[({A}_{\mathrm{cal}}^{\mathrm{T}}\left(I-{A}_{\mathrm{unx}}{A}_{\mathrm{unx}}^{+}\right){A}_{\mathrm{cal}})\ast ({B}_{\mathrm{cal}}^{T}\left(I-{B}_{\mathrm{unx}}{B}_{\mathrm{unx}}^{+}\right){{B}_{\mathrm{cal}}){]}^{-1}\}}_{nn}^{-1/2}$.

^{b}${\mathrm{SEN}}_{n}={l}_{n}\{[({A}_{\mathrm{cal}}^{\mathrm{T}}\left(I-{A}_{\mathrm{unx}}{A}_{\mathrm{unx}}^{+}\right){A}_{\mathrm{cal}})\ast ({B}_{\mathrm{cal}}^{T}\left(I-{B}_{\mathrm{unx}}{B}_{\mathrm{unx}}^{+}\right){{B}_{\mathrm{cal}}){]}^{-1}\}}_{nn}^{-1/2}$, A and B are the obtained normalized matrixes from the decomposition of ATLD or ATLD-MCR algorithm, subscript n identifies a particular analyte of interest, l

_{n}represents the total response signal for nth component at unit concentration.

^{c}${\mathrm{LOD}}_{n}=3.3{({\mathrm{SEN}}_{n}^{-2}{\sigma}_{x}^{2}+{h}_{0}{\mathrm{SEN}}_{n}^{-2}{\sigma}_{x}^{2}+{h}_{0}{\sigma}_{y\mathrm{cal}}^{2})}^{1/2}$.

^{d}${\mathrm{LOQ}}_{n}=10{({\mathrm{SEN}}_{n}^{-2}{\sigma}_{x}^{2}+{h}_{0}{\mathrm{SEN}}_{n}^{-2}{\sigma}_{x}^{2}+{h}_{0}{\sigma}_{y\mathrm{cal}}^{2})}^{1/2}$, σx denotes the standard deviation of analyte predicted concentration in three different unspiked samples. h0 is the value for the leverage in blank sample.

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## Share and Cite

**MDPI and ACS Style**

Chen, J.-C.; Wu, H.-L.; Wang, T.; Dong, M.-Y.; Chen, Y.; Yu, R.-Q.
High-Performance Liquid Chromatography–Diode Array Detection Combined with Chemometrics for Simultaneous Quantitative Analysis of Five Active Constituents in a Chinese Medicine Formula Wen-Qing-Yin. *Chemosensors* **2022**, *10*, 238.
https://doi.org/10.3390/chemosensors10070238

**AMA Style**

Chen J-C, Wu H-L, Wang T, Dong M-Y, Chen Y, Yu R-Q.
High-Performance Liquid Chromatography–Diode Array Detection Combined with Chemometrics for Simultaneous Quantitative Analysis of Five Active Constituents in a Chinese Medicine Formula Wen-Qing-Yin. *Chemosensors*. 2022; 10(7):238.
https://doi.org/10.3390/chemosensors10070238

**Chicago/Turabian Style**

Chen, Jun-Chen, Hai-Long Wu, Tong Wang, Ming-Yue Dong, Yue Chen, and Ru-Qin Yu.
2022. "High-Performance Liquid Chromatography–Diode Array Detection Combined with Chemometrics for Simultaneous Quantitative Analysis of Five Active Constituents in a Chinese Medicine Formula Wen-Qing-Yin" *Chemosensors* 10, no. 7: 238.
https://doi.org/10.3390/chemosensors10070238