Materials and reagents
5-Hydroxymethylfurfural (5-HMF, CAS: 67-47-0, purity > 99%), NƐ-(carboxymethyl)lysine (CML, CAS: 5746-04-3, purity > 99%) and NƐ-(carboxyethyl)lysine (CEL, CAS: 5746-03-2, purity > 99%) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol of HPLC grade were obtained from Fisher-Scientific (Waltham, MA, USA). Ultrapure water was obtained from a Millipore Milli-Q Advantage A10 system (Millipore, Bedford, USA). Chinese medicine injections were purchased from an ordinary pharmacy.
Determination of 5-HMF
A stock solution of 5-HMF was prepared at a concentration of 0.15 mg/mL, which was further diluted with 10% methanol to a series of calibration standards with concentrations of 0.15, 2.34, 9.38, 37.5, and 75 µg/mL. Quality control (QC) samples were also diluted to concentrations of 0.15, 9.38, and 75 µg/mL. A total of 200 µL of each Chinese medicine injection was diluted 10-fold with 10% methanol prior to HPLC analysis.
HPLC was conducted to determine the content of 5-HMF in different Chinese medicine injections. The separation was performed on a Hypersil ODS2 column (250 mm×4.6 mm, 5 µm, Waters Corp., Milford, MA, USA), and the column temperature was set at 30°C.The mobile phase was composed of water (solvent A) and methanol (solvent B). Gradient elution began with 5% solvent B for 5 min, followed by a linear gradient of 5-15% solvent B from 5 min to 15 min, and then returning to a linear gradient of 15%-5% solvent B from 15 min to 20 min. The flow rate was 1.0 mL/min, and the injection volume was 20 µL.
Method validation was conducted according to the US Food and Drug Administration guidelines [33], and method validation included selectivity, linearity, accuracy, precision, recovery and stability determination.
The selectivity of the method was determined by evaluating 6 blank reagent injections (10% methanol). The linearity was determined by using calibration standard samples at concentrations of 0.15, 2.34, 9.38, 37.5, and 75 µg/mL, and each level was independently injected 3 times. Calibration curves were constructed by plotting the average peak areas (y) of each sample against the calibration concentrations (x). The limits of detection (LODs) and quantification (LOQs) were evaluated by calculating the concentrations corresponding to signal-to-noise (S/N) ratios of 3 and 10, respectively [34]. The intra- and inter-day precision and accuracy were determined by measuring the concentrations of QC samples and were evaluated by the relative standard deviation (RSD). The stability of 5-HMF was determined by evaluating QC samples under three conditions, including three freeze-thaw cycles (-80°C), storage at room temperature for 4 h, storage at 4°C for 1 d, and storage at -20°C for 21 d. The recovery was assessed by calculating the ratios of concentrations between analytes in the QC samples and those in samples with known concentrations.
Determination of CML and CEL
A stock solution of CML and CEL was prepared with each at a concentration of 1 mg/mL, which was diluted to working solutions at a series of concentrations of 0.078, 0.313, 1.25, 5, 20, and 640 ng/mL. Two hundred microlitres of a Chinese medicine injection was measured and diluted 10-fold with ultrapure water, and 2 µL was injected into the UPLC system for analysis.
UPLC-MS/MS system was employed to analyse the concentrations of CML and CEL in Chinese medicine injections. The separation was achieved with a CORTECS HILIC UPLC column (2.1 mm×50 mm, 1.6 µm, Waters Corp., Milford, MA, USA) maintained at 40°C. Gradient elution was performed with water (solvent A) and acetonitrile (solvent B) with the following programme: 0-1 min (100% B), 1-2.5 min (100-60% B), 2.5-4 min (60% B), and 4-6 min (100% B). The flow rate was 0.3 mL/min, and the injection volume was 2 µL. quantification of CML and CEL were performed by using standards and by reference to an external standard calibration curve. Data are reported as means±SD of triplicate experiments.
Mass spectrometric detection was achieved on a Waters Xevo TQ mass spectrometer (Waters Corp., Milford, MA, USA) with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. MS detection was performed in positive ionization mode. The parent ions for CML and CEL were observed at m/z 205 and 219, respectively. CML and CEL were fragmented to produce ion signals at m/z 130.14→84.10 and m/z 130.15→84.10. Data acquisition was accomplished with Masslynx software version 4.1 (Waters Corp., Milford, MA, USA).
The method validation conducted as outlined in the description of “Determination of 5-HMF”. In brief, 6 blank reagent samples (ultrapure water) were used to measure the selectivity. The intra-/inter-day precision and accuracy were calculated using calibration standards at 0.078, 1.25 and 20 ng/mL. The precision was determined by the RSD, and the accuracy was expressed as RE. Recovery was determined by comparing the peak area ratios of samples spiked with calibration standards and the original amount. The stability of the working solution for CML and CEL was evaluated under three freeze-thaw cycles (-80°C), at room temperature for 4 h, at 4°C for 1 d, and at -20°C for 21 d.