Most ReadThe components of biological samples are complex, and the concentration range of target analytes varies significantly. Therefore, sample pretreatment is particularly important for subsequent analysis. With the continuous development of analytical techniques, the requirements for sample pretreatment are also increasing. Pretreatment technologies are evolving towards microscale, time-saving, high-efficiency, online, and environmentally friendly directions to meet the requirements of complex matrices and trace analysis. Online pretreatment technologies integrate sample pretreatment steps and subsequent detection processes directly into an automated system, which can reduce steps and errors, improve analysis efficiency and sensitivity, and achieve automatic, rapid, and efficient analysis of target compounds. This paper reviews the online pretreatment technologies for biological samples in China and abroad in the past 10 years, offering valuable insights to guide future research and innovation in the realm of biological sample pretreatment.
Objective: To identify and analyze the chemical components of Bufei Jianpi formula by ultra-high performance liquid chromatography-coupled with high-resolution mass spectrometry (UPLC-HRMS). Methods: A Hypersil GOLD (2.1 mm×100 mm, 2.6 μm) column was used. Methanol (A)-0.1% formic acid water (B) was used as the mobile phase with gradient elution, the flow rate was 0.2 mL · min-1, and the column temperature was 30 ℃.The mass spectrometry data were collected using the Full MS/dd-MS2 scanning mode of positive and negative ions, and the characteristic fragment ion peak information was analyzed by compound discoverer. Combined with Chemspider, mzCloud and other databases and existing reports of relevant chemical composition information,the chemical components of Bufei Jianpi formula was analyzed by using the cracking prediction of Mass Frontier and its cracking rule. Results: A total of 221 compounds were identified from Bufei Jianpi formula, including 65 flavonoids, 40 phenylpropanoids, 21 terpenoids, 16 alkaloids and 79 other compounds. Conclusion: UPLC-Orbitrap Fusion Lumos Tribrid-MS can quickly identify the chemical components of Bufei Jianpi formula and qualitatively analyze the material basis of Bufei Jianpi formula, which can be used for the quality control of Bufei Jianpi formula.
Objective: To develop an inductively coupled plasma mass spectrometry(ICP-MS) method for the determination of Cd, Pb, As, Hg, Co, V, Ni and Se in the crude drug of lanthanum carbonate. Methods: Samples were diluted with 3% nitric acid and directly injected. The matrix effect was eliminated by standard addition method and matrix matching method. Lanthanum oxide was added into the standard solution as the matrix.Compare the linearity, repeatability, accuracy, quantification limit, detection limit and sample determination results of the two methods by methodological validation and sample determination. Results: The linear ranges of ICP-MS standard addition method and matrix matching method were 0-30 ng · mL-1, the correlation coefficients for all elemental impurities were good(r>0.998). Limits of detection were between 0.003 9-0.22 ng · mL-1 and 0.009 1-0.72 ng · mL-1. The RSD of repeatability(n=6) were between 1.5%-4.9% and 5.1%-6.9%. The recoveries were between 87.0%-98.3% and 83.4%-114.4%. The contents of 8 elemental impurities in the crude drug of lanthanum carbonate were basically same. Conclusion: The established two methods can effectively eliminate the matrix effect. They were both simple, rapid, sensitive, accurate and applicable for the elemental impurity control of lanthanum carbonate.
To establish an in vitro permeation method for crisaborole ointment and to provide a reference for the bioequivalence of crisaborole ointment.
The HPLC method was used to determine the permeability of crisaborole ointment, and the vertical Franz diffusion pool method was used to study the release characteristics of the ointment in vitro. The chromatographic column was Thermo BDS Hypersil-C18(150 mm×4.6 mm, 5 μm), with 0.1% phosphoric acid solution-acetonitrile (60：40) as the mobile phase, flow rate of 1.5 mL·min-1, column temperature of 30 ℃, detection wavelength of 254 nm, and injection volume of 10 μL.
The peak shape of crisaborole in the chromatogram of the test sample was good. There was no peak at the position of crisaborole in the negative sample chromatogram, which did not interfere with the determination. The linear relationship of crisaborole was good within the range of 0.026-21.02 μg·mL-1 (r≥0.999). In six repeated experiments, the mean maximum transmittance (Jmax) of the self-made product and the reference formulation were compared (odds ratio: 0.94). Simultaneously calculate the confidence interval for Jmax, with a 90% confidence interval ranging from 87.9% to 102.0%. The mass conservation percentage of each sample was 94.1%-101.1%(n=12). The reference solution was left at room temperature for 26 h, with a peak area RSD of 0.44%. The self-made product and reference preparation were left in a water bath at room temperature and 32 ℃ for 26 h, with peak area RSD ranging from 0.78% to 1.1%, indicating good stability of the solution. For the three batches of samples tested, the ratio of the mean Jmax values of the self-made product and the reference formulation was within the range of 0.92-0.97. Simultaneously calculate the confidence interval for Jmax, with a 90% confidence interval ranging from 84.8%-100.7%.
This method is simple and convenient to operate, with high selectivity, strong exclusivity and high accuracy.
Objective: To establish a selective culture medium screening method for counting viable bacteria from multi-strain microecological preparation based on gene and metabolic pathway analysis, in order to solve the problem of mutual interference in the measurement of counting viable bacteria. Methods: By analyzing the genomic scale metabolic network, the antibiotic sensitivity and resistance of Bacillus cereus, Enterococcus faecalis,Lactobacillus acidophilus, and Bifidobacterium infantis were quickly predicted. Based on the prediction results,suitable types and concentrations of antibiotics with resistance differences among the four microorganisms were screened and added to the culture medium, effectively inhibiting the growth of interfering microorganisms and ensuring accurate and effective counting results. Simultaneously, conventional drug sensitivity experiments results were used to ensure and compare with this method. Results: In the process of viable counting for multi-strain microecological preparation, this method effectively eliminated the interference of Enterococcus faecalis on the counting of Lactobacillus acidophilus live bacteria and the interference of Lactobacillus acidophilus and Enterococcus faecalis on the counting of Bifidobacterium infant, and the counting results were accurate and reliable of each microorganism. Conclusion: The method has strong foresight and specificity. Genomic analysis and metabolic pathway analysis can effectively predict microbial antibiotic resistance, and experimental design based on the predicted results will greatly accelerate experimental efficiency, making the results more predictive and accurate,and has broad application prospects in selective culture medium screening for counting viable bacteria from multi-strain microecological preparation.
Objective: To clarify the differences between the “Room temperature” definitions in the European Pharmacopoeia and the Chinese Pharmacopoeia, to emphasize that storage temperature was closely related to the design of long-term stability studies and product labeling, and to investigate the effects of storage temperature variations on the critical quality attributes (CQAs) of terlipressin for injection, so as to raise awareness among generic drug manufacturers regarding this critical issue. Methods: Given the discrepancies in storage temperature recommendations across different manufacturers’ product inserts, the original drug’s long-term stability studies and storage conditions were traced. Validated methods for related substances and polymer content were employed to assess the impact of temperature differences on the product’s CQAs. Results: Samples stored at 30 ℃exhibited a significantly higher increase in related substances compared to those stored at 15 ℃. In one manufacturer’s product, polymer levels exceeded specification limits within just five months of storage at 30 ℃. The divergence among manufacturers stems from some companies misinterpreting the original drug’s labeling by directly translating without considering the differences between Chinese and European Pharmacopoeias. Conclusion: Storage temperature has a significant impact on the levels of related substances and polymer content in terlipressin for injection. To ensure product quality, the storage temperature in the labeling should be restricted to “not exceeding 25 ℃.”
To develop a flow-through cell method for the dissolution test of omega-3-acid ethyl ester 90 soft capsules and compare the dissolution behaviors from different manufacturers.
The medium (surfactant and its concentration, pH, dosage of pepsin), flow rate and system mode (closed versus open) were investigated. The samples were collected at the specified time and determined by HPLC. The similarity of the dissolution curves between generic drugs and reference listed drug was evaluated by similarity factor (f2).
A closed-loop mode of flow-through cell apparatus was employed, with 0.01 mol·L-1 hydrochloric acid solution containing 4.0% Triton X-100 as the dissolution medium, and the flow rate was 2.0 mL·min-1. The dissolution curves of the samples that have passed consistency evaluation are similar to those of the reference and the samples produced by enterprise in the declaration stage are partly similar. The method has effective distinguish ability for product quality and different prescriptions.
The newly established method can be used for the quality control of omega-3-acid ethyl ester 90 soft capsules, and can provide references for further consistency evaluation and the dissolution method development of lipid-filled soft gelatin capsule (SGC).
Objective: To establish an HPLC method for the simultaneous determination of chlorogenic acid,puerarin, 3’-methoxy puerarin, puerarin apioside, liquiritin, forsythoside A, 3,5-O-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid, forsythin, honokiol and magnolol in Jinlian Quwen capsules. Methods: The analysis was performed on a Waters AtlantisTM T3 (150 mm×4.6 mm, 3 μm) column, and the mobile phase was comprised of acetonitrile-0.1% phosphoric acid, with the flow rate of 1.0 mL · min-1 in a gradient elution manner. The column temperature was set at 35 ℃. The injection volume was 4 μL and the UV detection wavelengths were set at 225 nm (detecting forsythin), 250 nm (detecting puerarin, 3’-methoxy puerarin and puerarin apioside), 276 nm(detecting liquiritin), 290 nm (detecting honokiol and magnolol) and 325 nm (detecting chlorogenic acid,3,5-O-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and forsythoside A). Results: All the 11 constituents showed good linearity within their detection ranges (r≥0.999 5), whose average recoveries (n=6) were 100.1%-108.1%, with the RSDs of 2.8%-4.1%. The content ranges of 11 components in three batches of samples were 7.505-7.606 mg · g-1, 3.485-3.920 mg · g-1, 0.969-1.068 mg · g-1, 0.837-0.955 mg · g-1, 1.009-1.436 mg · g-1,3.037-3.602 mg · g-1, 5.259-5.371 mg · g-1, 0.931-1.012 mg · g-1, 1.428-2.053 mg · g-1, 0.939-1.018 mg · g-1 and 2.744-2.827 mg · g-1, respectively. Conclusion: The method is simple, sensitive and reliable. It can be used as a reference for the establishment of quality standard of Jinlian Quwen capsules.
Objective: To establish an ultra-performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-QQQ MS/MS) method for the simultaneous determination of 14 active components in Qiyu Sanlong decoction,including L-argnine, monotropein, deacetyl asperulosidic acid, rutin, peimisine, calycosin-7-O-β-D-glucoside,caffeic acid, solasonine, solamargine, p-coumaric acid, ferulic acid, calycosin, astragaloside Ⅳ and astragaloside Ⅰ. Methods: The chromatographic separation experiment was performed on an ACQUITY UPLC BEH C18 column(2.1 mm×100 mm, 1.7 μm), with gradient elution of 0.1% formic acid aqueous solution (A)-acetonitrile (B) as mobile phase at the flow rate of 0.2 mL · min-1, injection volume of 5 μL, and column temperature of 35 ℃. The ion source was an electrospray ionization source (ESI), the scanning mode was simultaneous scanning of positive and negative ions, and the monitoring mode was multiple reaction monitoring. Results: The 14 active components revealed good linearity within their respective ranges (r>0.995 0), RSDs of precision and repeatability were below 2%, RSDs of stability were below 3%, and the average recoveries ranged from 88.4% to 108.5% with the RSDs ranging from 0.020%to 3.6%. The content ranges of the aforementioned 14 components in 10 batches of self-prepared Qiyu Sanlong decoction were as follows (in μg · g-1): 667.28-785.78, 165.72-197.27, 196.32-275.60, 17.60-26.52, 4.68-10.75,279.12-388.05, 26.00-47.57, 385.52-442.77, 288.00-358.82, 629.88-839.02, 86.67-125.83, 51.58-65.83, 25.50-37.53, and 55.50-76.13. Conclusion: The UPLC-QQQ MS/MS method established in this study is rapid, accurate,sensitive and repeatable, which can provide a reference for the quality control of Qiyu Sanglong decoction.
To establish a high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q TOF MS) method for the simultaneous analysis of illegal 4 anti-obesity small molecule drugs and 4 glucagon-like peptide-1 (GLP-1) peptide additives.
The samples were extracted by ultrasound using 50% acetonitrile as the extraction solvent. After centrifugation, the supernatant was taken and separated using an Agilent EC-C18 chromatographic column (150 mm×3.0 mm, 2.7 μm). Acetonitrile (0.1% formic acid) and water (0.1% formic acid) were used as mobile phases, with gradient elution at a flow rate of 0.3 mL·min-1 and column temperature of 40 ℃. Adopting positive ion full scanning and target ion secondary fragment scanning methods, with a fragmentation voltage of 150 V. The scanning range of the primary mass spectrometry was m/z 100-3 200, and the scanning range of the secondary mass spectrometry was m/z 50-3 200, with a scanning speed of 1 mass spectrum per second. Establish a data spectral library based on the chromatographic retention time, primary mass spectrometry, and secondary mass spectrometry information of the reference standard, and confirmed the structure through database comparison.
The screening detection limit for peptides was 0.5 µg·mL-1, while small molecular drugs was 0.05 µg·mL-1. The recoveries were in the range of 79.4% to 115.8%，with the relative standard deviations of 0.21% to 9.7%. Using this method, 20 batches of anti-obesity drugs were tested, in which semaglutide was identified in 4 samples and sibutratmine was identified in 1 batche.
Compared with the complementary method No. 2012005 by the China Food and Drug Administration, the method established in this study can simultaneously analyze small and large molecules (the relative molecular mass<5 000), featuring high efficiency and accuracy.
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