Guan-Yuan Chen

Simultaneous quantification of antimicrobial agents for multidrug-resistant bacterial infections in human plasma by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Antibiotic-resistant bacterial infection is one of the most serious clinical problems worldwide. Vancomycin, teicoplanin, daptomycin, and colistin are glycopeptide and lipopeptide antibiotics that are frequently used to treat multidrug-resistant bacterial infections. Therapeutic drug monitoring is recommended to ensure both safety and efficacy and to improve clinical outcomes. This study developed a fast, simple, and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of the concentrations of these four drugs in human plasma. The sample preparation process includes a simple protein denaturation step using acetonitrile, followed by an 11-fold dilution with 0.1% formic acid. Eight target peaks for the four drugs can be analyzed within 3 min using a Kinetex™ 2.6 μm C18 column. The mass spectrometry parameters were optimized, and two transitions for each target peak were used for multiple reaction monitoring, which provided high sensitivity and specificity. The UHPLC-MS/MS method was validated over clinical concentration ranges. The intra-day and inter-day precisions for the ratio of the peak area of each analyte to the peak area of the internal standard were all below 12.7 and 14.7% relative standard deviations, respectively. The accuracy at low, medium, and high concentrations of the eight target peaks was between 89.3 and 110.7%. The standard curves for the analytes were linear and had coefficients of determination higher than 0.997. The limits of detection were all below 70 ng mL(-1). The use of this method to analyze patient plasma samples confirmed that it is effective for the therapeutic drug monitoring of these four drugs and can be used to improve the therapeutic efficacy and safety of treatment with antibiotics.

Using a postcolumn-infused internal standard for correcting the matrix effects of urine specimens in liquid chromatography–electrospray ionization mass spectrometry

Matrix effects (MEs) are a major problem affecting the quantitative accuracy of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) when analyzing complicated samples. While analyzing urine specimens, the wide diversity of endogenous materials and different urine concentrations may result in inaccurate quantification. In this study, we propose a postcolumn-infused internal standard (PCI-IS) strategy for universal correction of MEs in urine specimens. MEs can be effectively corrected by dividing the target analyte signal intensity by the PCI-IS intensity. To evaluate the performance of PCI-IS, we used 6 benzodiazepine (BZD) drugs in 5 different concentrations of urine matrixes as a test model. The divergence of the BZD drug signal responses in 5 different urine matrixes was expressed using their respective coefficients of variation (CV) to evaluate the efficiency of using PCI-IS in correcting matrix effects. The CV of the BZD drug signal intensities in these 5 different concentrations of the urine matrixes were reduced from 10 to 30% to less than 10% when the PCI-IS correction method was employed. When the PCI-IS method was used to correct the 6 BZDs in 25 real human urine samples, over 90% of the test results exhibited quantification errors of less than 20%, and all of the test results had quantification errors of less than 30%. These results demonstrate that the PCI-IS method can resolve the problem of inaccurate quantification that arises from the diversity of urine specimens. The PCI-IS method is particularly useful for clinical analysis or forensic toxicology to improve the quantification accuracy in an economical way.

True ion pick (TIPick): a denoising and peak picking algorithm to extract ion signals from liquid chromatography/mass spectrometry data

Liquid Chromatography-Time of Flight Mass Spectrometry has become an important technique for toxicological screening and metabolomics. We describe TIPick a novel algorithm that accurately and sensitively detects target compounds in biological samples. TIPick comprises two main steps: background subtraction and peak picking. By subtracting a blank chromatogram, TIPick eliminates chemical signals of blank injections and reduces false positive results. TIPick detects peaks by calculating the S(CC(INI)) values of extracted ion chromatograms (EICs) without considering peak shapes, and it is able to detect tailing and fronting peaks. TIPick also uses duplicate injections to enhance the signals of the peaks and thus improve the peak detection power. Commonly seen split peaks caused by either saturation of the mass spectrometer detector or a mathematical background subtraction algorithm can be resolved by adjusting the mass error tolerance of the EICs and by comparing the EICs before and after background subtraction. The performance of TIPick was tested in a data set containing 297 standard mixtures; the recall, precision and F-score were 0.99, 0.97 and 0.98, respectively. TIPick was successfully used to construct and analyze the NTU MetaCore metabolomics chemical standards library, and it was applied for toxicological screening and metabolomics studies.

Metabolomic Analysis of Complex Chinese Remedies: Examples of Induced Nephrotoxicity in the Mouse from a Series of Remedies Containing Aristolochic Acid

Aristolochic acid nephropathy is caused by aristolochic acid (AA) and AA-containing herbs. In traditional Chinese medicine, a principle called “Jun-Chen-Zou-Shi” may be utilized to construct a remedial herbal formula that attempts to mitigate the toxicity of the main ingredient. This study used Bu-Fei-A-Jiao-Tang (BFAJT) to test if the compound remedy based on a principle of “Jun-Chen-Zou-Shi” can decrease the toxicity of AA-containing herbs. We compared the three toxicities of AA standard, Madouling (an Aristolochia herb), and a herbal formula BFAJT. AA standard was given for BALB/c mice at a dose of 5 mg/kg bw/day or 7.5 mg/kg bw/day for 10 days. Madouling and BFAJT were given at an equivalence of AA 0.5 mg/kg bw/day for 21 days. Nephrotoxicity was evaluated by metabolomics and histopathology. The urinary metabolomics profiles were characterized by 1H NMR spectroscopy. The spectral data was analyzed with partial least squares discriminant analysis, and the significant differential metabolites between groups were identified. The result showed different degrees of acute renal tubular injuries, and metabolomics analysis found that the kidney injuries were focused in proximal renal tubules. Both metabolomics and pathological studies revealed that AA standard, Madouling, and BFAJT were all nephrotoxicants. The compositions of the compound remedy did not diminish the nephrotoxicity caused by AA.

Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis

CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes.

Quantification of target analytes in various biofluids using a postcolumn infused-internal standard method combined with matrix normalization factors in liquid chromatography–electrospray ionization mass spectrometry

Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has become one of the most widely used methods in pharmaceutical laboratories. Although LC-ESI-MS provides high sensitivity and high specificity for quantifying target analytes in complicated biofluids, the associated severe matrix effects (MEs) generally result in large quantification errors. Here, we propose a novel strategy for correcting MEs in various biofluids using a postcolumn infused-internal standard (PCI-IS) method in combination with matrix normalization factors (MNFs). We used the MNFs to normalize the encountered MEs in various biofluids to the MEs encountered in standard solutions. The use of a postcolumn infused-internal standard also corrects the MEs for individual samples. When using the PCI-IS method in combination with MNFs, the calibration curve generated from standard solutions can be applied to quantify the target analytes in various biofluids. We applied this new approach to quantify etoposide and etoposide catechol in plasma and CSF. The accuracy of the test results showed that over 93% of the data have quantification errors less than 20% and that 99% of the data have quantification errors less than 30%. The successful application of this method to evaluate real clinical samples revealed that our proposed MNFs in combination with the PCI-IS method largely simplifies the entire method development and validation processes, saves a great deal of time and cost without sacrificing quantification accuracy, and provides a simple means of quantifying target analytes in various biofluids. This method will be particularly useful in fields in which the same target analytes need to be quantified in various types of matrices, including bioanalysis, forensic toxicology, environmental studies, and food safety control.

Estimation and Correction of the Blood Volume Variations of Dried Blood Spots Using a Postcolumn Infused-Internal Standard Strategy with LC-Electrospray Ionization-MS

Dried blood spots (DBSs) have had a long history in disease screening in newborns but have gained attention in recent years in the medical care of adults because of the growing importance of personalized medicine. DBSs have several advantages, such as easy transportation, cost-effectiveness, and minimally invasive biological sampling. There are two strategies to process DBS samples. One method takes a fixed diameter of subsample, and another requires the extraction of the whole spot. The whole-spot extraction method is less affected by hematocrit-caused errors, but it requires calibration of the blood volume. We propose a novel strategy using a postcolumn infused-internal standard (PCI-IS) method with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for estimating and correcting blood volume variations on the DBS cards. By using PCI-IS to measure the extent of ion suppression in the first ion suppression zone in the chromatogram, the blood volume on the DBS cards can be calculated and further calibrated. We used reference blood samples with different volumes (5 to 25 μL) to construct a calibration curve between the blood volume and the extent of ion suppression. The calibration curve was used to estimate the blood volume on the DBS cards collected from 6 volunteers, with 5 designated volumes from each volunteer. The estimation accuracy of the PCI-IS method was between 74.5% and 120.3%. The validated PCI-IS method was used to estimate and calibrate blood volume variation and also to quantify the voriconazole concentration for 26 patients undergoing voriconazole therapy. A high correlation was found for the quantification results between the DBS samples and the conventionally used plasma samples (r = 0.97). The PCI-IS method was demonstrated to be a simple and accurate method for estimating and calibrating the blood volume variation on DBS cards, which greatly facilitates using the DBS method for therapeutic drug monitoring (TDM) for improving the efficacy and safety of drug therapy.

Quantification of endogenous metabolites by the postcolumn infused-internal standard method combined with matrix normalization factor in liquid chromatography-electrospray ionization tandem mass spectrometry

Quantification of endogenous metabolites has enabled the discovery of biomarkers for diagnosis and provided for an understanding of disease etiology. The standard addition and stable isotope labeled-internal standard (SIL-IS) methods are currently the most widely used approaches to quantifying endogenous metabolites, but both have some limitations for clinical measurement. In this study, we developed a new approach for endogenous metabolite quantification by the postcolumn infused-internal standard (PCI-IS) method combined with the matrix normalization factor (MNF) method. MNF was used to correct the difference in MEs between standard solution and biofluids, and PCI-IS additionally tailored the correction of the MEs for individual samples. Androstenedione and testosterone were selected as test articles to verify this new approach to quantifying metabolites in plasma. The repeatability (n=4 runs) and intermediate precision (n=3 days) in terms of the peak area of androstenedione and testosterone at all tested concentrations were all less than 11% relative standard deviation (RSD). The accuracy test revealed that the recoveries were between 95.72% and 113.46%. The concentrations of androstenedione and testosterone in fifty plasma samples obtained from healthy volunteers were quantified by the PCI-IS combined with the MNF method, and the quantification results were compared with the results of the SIL-IS method. The Pearson correlation test showed that the correlation coefficient was 0.98 for both androstenedione and testosterone. We demonstrated that the PCI-IS combined with the MNF method is an effective and accurate method for quantifying endogenous metabolites.

Development and application of a comparative fatty acid analysis method to investigate voriconazole-induced hepatotoxicity.

BACKGROUND As fatty acids play an important role in biological regulation, the profiling of fatty acid expression has been used to discover various disease markers and to understand disease mechanisms. This study developed an effective and accurate comparative fatty acid analysis method using differential labeling to speed up the metabolic profiling of fatty acids. METHODS Fatty acids were derivatized with unlabeled (D0) or deuterated (D3) methanol, followed by GC-MS analysis. The comparative fatty acid analysis method was validated using a series of samples with different ratios of D0/D3-labeled fatty acid standards and with mouse liver extracts. RESULTS Using a lipopolysaccharide (LPS)-treated mouse model, we found that the fatty acid profiles after LPS treatment were similar between the conventional single-sample analysis approach and the proposed comparative approach, with a Pearsons correlation coefficient of approximately 0.96. We applied the comparative method to investigate voriconazole-induced hepatotoxicity and revealed the toxicity mechanism as well as the potential of using fatty acids as toxicity markers. CONCLUSIONS In conclusion, the comparative fatty acid profiling technique was determined to be fast and accurate and allowed the discovery of potential fatty acid biomarkers in a more economical and efficient manner.

Sensitive screening of abused drugs in dried blood samples using ultra-high-performance liquid chromatography-ion booster-quadrupole time-of-flight mass spectrometry

An increased rate of drug abuse is a major social problem worldwide. The dried blood spot (DBS) sampling technique offers many advantages over using urine or whole blood sampling techniques. This study developed a simple and efficient ultra-high-performance liquid chromatography-ion booster-quadrupole time-of-flight mass spectrometry (UHPLC-IB-QTOF-MS) method for the analysis of abused drugs and their metabolites using DBS. Fifty-seven compounds covering the most commonly abused drugs, including amphetamines, opioids, cocaine, benzodiazepines, barbiturates, and many other new and emerging abused drugs, were selected as the target analytes of this study. An 80% acetonitrile solvent with a 5-min extraction by Geno grinder was used for sample extraction. A Poroshell column was used to provide efficient separation, and under optimal conditions, the analytical times were 15 and 5min in positive and negative ionization modes, respectively. Ionization parameters of both electrospray ionization source and ion booster (IB) source containing an extra heated zone were optimized to achieve the best ionization efficiency of the investigated abused drugs. In spite of their structural diversity, most of the abused drugs showed an enhanced mass response with the high temperature ionization from an extra heated zone of IB source. Compared to electrospray ionization, the ion booster (IB) greatly improved the detection sensitivity for 86% of the analytes by 1.5-14-fold and allowed the developed method to detect trace amounts of compounds on the DBS cards. The validation results showed that the coefficients of variation of intra-day and inter-day precision in terms of the signal intensity were lower than 19.65%. The extraction recovery of all analytes was between 67.21 and 115.14%. The limits of detection of all analytes were between 0.2 and 35.7ngmL-1. The stability study indicated that 7% of compounds showed poor stability (below 50%) on the DBS cards after 6 months of storage at room temperature and -80°C. The reported method provides a new direction for abused drug screening using DBS.