A dilute-and-shoot flow-injection tandem mass spectrometry method for quantification of phenobarbital in urine.

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the gold standard of urine drug testing. However, current LC-based methods are time consuming, limiting the throughput of MS-based testing and increasing the cost. This is particularly problematic for quantification of drugs such as phenobarbital, which is often analyzed in a separate run because they must be negatively ionized. METHODS: This study examined the feasibility of using a dilute-and-shoot flow-injection method without LC separation to quantify drugs with phenobarbital as a model system. Briefly, a urine sample containing phenobarbital was first diluted by 10 times, followed by flow injection of the diluted sample to mass spectrometer. Quantification and detection of phenobarbital were achieved by an electrospray negative ionization MS/MS system operated in the multiple reaction monitoring (MRM) mode with the stable-isotope-labeled drug as internal standard. RESULTS: The dilute-and-shoot flow-injection method developed was linear with a dynamic range of 50-2000 ng/mL of phenobarbital and correlation coefficient > 0.9996. The coefficients of variation and relative errors for intra- and inter-assays at four quality control (QC) levels (50, 125, 445 and 1600 ng/mL) were 3.0% and 5.0%, respectively. The total run time to quantify one sample was 2 min, and the sensitivity and specificity of the method did not deteriorate even after 1200 consecutive injections. CONCLUSIONS: Our method can accurately and robustly quantify phenobarbital in urine without LC separation. Because of its 2 min run time, the method can process 720 samples per day. This feasibility study shows that the dilute-and-shoot flow-injection method can be a general way for fast analysis of drugs in urine. Copyright © 2016 John Wiley & Sons, Ltd.

A rapid LC-MS/MS method for quantification of CSUOH0901, a novel antitumor agent, in rat plasma.

CSUOH0901, a novel anticancer derivative of nimesulide, exhibits very promising anticancer activities in various cancer cell lines. In order to support further pharmacological and toxicological studies of this promising anticancer drug candidate, an LC-MS/MS method was developed and validated in accordance with the US Food and Drug Administration guidelines. The drug molecules were extracted from plasma samples by protein precipitation and then analyzed with LC-ESI-MS/MS. An excellent analyte separation was achieved using a phenomenex C18 column with a mobile phase of 90% methanol and 5 m m of ammonium formate. The validated linear dynamic range was between 0.5 and 100 ng/mL and the achieved correlation coefficient (r(2)) was >0.9996. The results of inter- and intra-day precision and accuracy were satisfactory, that is, <12% for accuracy and within ±5% for precision at a low and high quality control concentrations, respectively. In addition, the analyte and internal standard (JCC76) were found to be stable under the storage conditions at -20°C for about 2 months. Hence, the acquired results proved that the LC-ESI-MS/MS method developed is precise, accurate and selective for the quantification of CSUOH0901 in plasma, and can be used for pharmacokinetic studies.

Development and validation of a rapid LC-MS/MS method for the determination of JCC76, a novel antitumor agent for breast cancer, in rat plasma and its application to a pharmacokinetics study.

JCC76 is a novel nimesulide analog that selectively inhibits the human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cell proliferation and tumor progression. To support further pharmacological and toxicological studies of JCC76, a novel and rapid method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the quantification of the compound in rat plasma. A C18 column was used for chromatographic separation, and the mobile phase was aqueous ammonium formate (pH 3.7; 5 mm)-methanol (1:9, v/v) with an isocratic elution. With a simple liquid-liquid extraction procedure using the mixture of methyl tert-butyl ether-hexane (1:2, v/v), the mean extraction efficiency of JCC76 in rat plasma was determined as 89.5-97.3% and no obvious matrix effect was observed. This method demonstrated a linear calibration range from 0.3 to 100 ng/mL for JCC76 in rat plasma and a small volume of sample consumption. The intra- and inter-assay accuracy and precision were within ±10%. The pharmacokinetics of JCC76 was also profiled using this validated method in rats. In conclusion, this rapid and sensitive method has been proven to effectively quantify JCC76 for pharmacokinetics study.

An LC-ESI-MS method for the quantitative analysis of bile acids composition in fecal materials.

In this work, a liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated for quantification of bile acids in fecal materials. Co-eluting matrix impurities in fecal materials have been shown to greatly suppress the ionization of analytes in mass spectrometry, which is known as the matrix effect. To correct large quantitative errors caused by the matrix effect, we developed a scheme that combined the standard addition method with internal standard (SA-IS). The fecal sample pretreatment involved a single step of extraction with ethanol. Bile acids were separated using a Luna C(18) column (150 mm, 2 mm i.d., 5 µm) with gradient elution. The deprotonated analytes were detected in selective ion monitoring mode. Our results showed that, by using this method, the accuracy of quantification was significantly improved in comparison to the conventional internal standard method. The linearity, sensitivity, accuracy and precision of the method were within the range of 0.05-5 µmol/L. This SA-IS method was successfully applied to the analysis of bile acids in the samples collected from patients diagnosed with inflammatory bowel disease.

A fast, fully validated GC-MS method using a simplified pretreatment for the quantification of short and branched chain fatty acids in human stool.

The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism, and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acid) in stool. Therefore, a GC-MS method meeting this criterion was developed. A bias sampling study showed no statistical difference (p > 0.05) in analyte means when comparing 100 mg subsamples of homogenized to non-homogenized samples (n = 6, p values 0.153-0.910). Stool samples were homogenized, diluted with 80:20 water:methanol (v/v), and adjusted to a pH of 1.5-2.5. Samples were vortexed, centrifuged, and directly injected into the GC-MS using pulsed splitless injection offering twofold-to-threefold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses, or retention time shifts after 1,476 stool injections, while other columns failed before 361 injections. Intra- and inter-day accuracy for stool supernatant samples ranged from -10.21% to 8.88% and -13.25% to 9.91%, while intra- and inter-day precision ranged from 0.21% to 1.21% and 0.89% to 2.84% coefficient of variation (CV), respectively. This method demonstrates excellent linearity (0.9999-1.0000) and low limits of quantification (1.50-8.01 µM). Stool samples proved stable stored at -20°C up to 28 days, and recoveries ranged from 85.04% to 106.59%. Matrix effects in stool are non-significant determined by comparing standard and stool supernatant calibration curve slopes (p > 0.05).

A matrix of 3,4-diaminobenzophenone for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A new matrix of 3,4-diaminobenzophenone (DABP) was demonstrated to be advantageous in the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With DABP as a matrix, intact oligonucleotide ions can be readily produced with lower laser powers, resulting in better detection limits, less fragmentation and fewer alkali metal ion adducts compared with the results obtained with conventional matrices. Importantly, minimal fragmentation and fewer alkali metal ion adducts were seen even at low concentrations of oligonucleotides. It was also found that samples prepared with DABP are highly homogenous and therefore reducing the need for finding 'sweet' spots in MALDI. In addition, excellent shot-to-shot reproducibility, resolution and signal-to-noise ratio were seen with DABP as the matrix.

Detection of mononucleotide repeat sequence alterations in a large background of normal DNA for screening high-frequency microsatellite instability cancers.

PURPOSE: Mutations in mononucleotide repeat sequence (MRS) are good indicators of high-frequency microsatellite instability (MSI-H) cancers, but it has been a challenge to detect such mutations in a large background of wild-type DNA; as in this setting, PCR errors often generate false positive mutant alleles. In this study, we developed a general strategy, referred to as probe clamping primer extension-PCR (PCPE-PCR), to detect MRS alterations in a large background of wild-type DNA. EXPERIMENTAL DESIGN: In PCPE-PCR, genomic DNA is first subjected to PCPE, in which mutant single-strand DNA molecules are preferentially produced. Next, genomic DNA is removed to enrich for the mutant DNA fraction. Thereafter, PCR is carried out using the remaining single-strand DNA molecules as templates. Finally, the PCR products are analyzed to reveal the MSI-H status. In this study, the sensitivity of this new method was first examined by spiking mutant DNA into wild-type DNA at specific ratios followed by studying whether this method is applicable to fecal DNA testing. RESULTS: We showed that PCPE-PCR could detect both mutated BAT26 and transforming growth factor-beta-RII (A)10 markers in the presence of 500-fold excess of normal DNA and that as few as three copies of mutated DNA could be detected. In addition, we showed that this technology could detect MSI-H colorectal cancer by fecal DNA analysis. CONCLUSION: PCPE-PCR is sensitive. In addition, PCPE-PCR is simple and amendable to a cost-effective and high-throughput screening operation. This technology may be applicable to noninvasive screening of MSI-H cancer.

Detection of tumor mutations in the presence of excess amounts of normal DNA.

Mutations are important markers in the early detection of cancer. Clinical specimens such as bodily fluid samples often contain a small percentage of mutated cells in a large background of normal cells. Thus, assays to detect mutations leading to cancer need to be highly sensitive and specific. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening. Here we describe a screening method, termed PPEM (PNA-directed PCR, primer extension, MALDI-TOF), that addresses these needs more effectively than do existing methods. DNA samples are first amplified using peptide nucleic acid (PNA)-directed PCR clamping reactions in which mutated DNA is preferentially enriched. The PCR-amplified DNA fragments are then sequenced through primer extension to generate diagnostic products. Finally, mutations are identified using matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method can detect as few as 3 copies of mutant alleles in the presence of a 10,000-fold excess of normal alleles in a robust and specific manner. In addition, the method can be adapted for simultaneous detection of multiple mutations and is amenable to high-throughput automation.

Genotyping single-nucleotide polymorphisms by matrix-assisted laser desorption/ionization time-of-flight-based mini-sequencing.

Currently, there is a critical need to develop high-throughput, low-cost, and accurate methods for genotyping of single-nucleotide polymorphisms (SNPs). The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometrically based technique represents a new promising approach to SNP analysis. We have developed a new MALDI-TOF-based mini-sequencing assay, termed "VSET," for genotyping of SNPs. In this assay, specific fragments of genomic DNA containing the SNP site(s) are first amplified, followed by mini-sequencing in the presence of three ddNTPs and the fourth nucleotide in the deoxy form. In this way, the primer is extended by only one base from one allele, whereas it is typically extended by two bases from another allele. The products are then analyzed using MALDI-TOF mass spectrometry. The genotype of the SNP site is identified based on the number of nucleotides added.

Carbon nanotubes as adsorbent of solid-phase extraction and matrix for laser desorption/ionization mass spectrometry.

A method with carbon nanotubes functioning both as the adsorbent of solid-phase extraction (SPE) and the matrix for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to analyze small molecules in solution has been developed. In this method, 10 microL suspensions of carbon nanotubes in 50% (vol/vol) methanol were added to the sample solution to extract analytes onto surface of carbon nanotubes because of their dramatic hydrophobicity. Carbon nanotubes in solution are deposited onto the bottom of tube with centrifugation. After removing the supernatant fluid, carbon nanotubes are suspended again with dispersant and pipetted directly onto the sample target of the MALDI-MS to perform a mass spectrometric analysis. It was demonstrated by analysis of a variety of small molecules that the resolution of peaks and the efficiency of desorption/ionization on the carbon nanotubes are better than those on the activated carbon. It is found that with the addition of glycerol and sucrose to the dispersant, the intensity, the ratio of signal to noise (S/N), and the resolution of peaks for analytes by mass spectrometry increased greatly. Compared with the previously reported method by depositing sample solution onto thin layer of carbon nanotubes, it is observed that the detection limit for analytes can be enhanced about 10 to 100 times due to solid-phase extraction of analytes in solution by carbon nanotubes. An acceptable result of simultaneously quantitative analysis of three analytes in solution has been achieved. The application in determining drugs spiked into urine has also been realized.

Using oxidized carbon nanotubes as matrix for analysis of small molecules by MALDI-TOF MS.

Oxidized carbon nanotubes are tested as a matrix for analysis of small molecules by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Compared with nonoxidized carbon nanotubes, oxidized carbon nanotubes facilitate sample preparation because of their higher solubility in water. The matrix layer of oxidized carbon nanotubes is much more homogeneous and compact than that of nonoxidized carbon nanotubes. The efficiency of desorption/ionization for analytes and the reproducibility of peak intensities within and between sample spots are greatly enhanced on the surface of oxidized carbon nanotubes. The advantage of the oxidized carbon nanotubes in comparison with alpha-cyano-4-hydroxycinnamic acid (CCA) and carbon nanotubes is demonstrated by MALDI-TOF-MS analysis of an amino acid mixture. The matrix is successfully used for analysis of synthetic hydroxypropyl beta-cyclodextrin, suggesting a great potential for monitoring reactions and for product quality control. Reliable quantitative analysis of jatrorrhizine and palmatine with a wide linear range (1-100 ng/mL) and good reproducibility of relative peak areas (RSD less than 10%) is achieved using this matrix. Concentrations of jatrorrhizine (8.65 mg/mL) and palmatine (10.4 mg/mL) in an extract of Coptis chinensis Franch are determined simultaneously using the matrix and a standard addition method.

A Simple and Rapid LC-MS/MS Method for the Determination of BMCL26 a Novel Anti-Parasitic Agent in Rat Plasma.

BMCL26 is a potential drug derived from nimesulide, which has exhibited the substantial anti-parasitic activity in various cell lines. To conduct various pharmacological and toxicological properties of this drug, we developed and validated a rapid LC-MS/MS method for its quantification in accordance with the FDA guidelines. Protein precipitation with 0.1% formic acid in acetonitrile was used to extract the analytes along with the internal standard (JCC76) from rat plasma. It was found that the calibration curve of the method had an excellent linearity (r2 ≥ 0.9993) for the analyte concentration ranging from 0.5 to 100 ng/mL with acceptable inter- and intra-assay, precision, accuracy and stability. The matrix effect and extraction recovery were in the range of 101.30-110.10% and 90.16- 105.00%, respectively. This LC-MS/MS method is simple and rapid and can be used in the future pharmaceutical studies of BMCL26.

Sumoylation of vimentin354 is associated with PIAS3 inhibition of glioma cell migration.

The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet the molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Over-expression of PIAS3 effectively changes cell shape and inhibits GBM cell migration. We focused on the molecular target(s) of PIAS3 stimulated sumoylation, which play an important role in the inhibition of GBM cell motility. Here we report, through the immunoprecipitation with SUMO1 antibody, followed by proteomic analysis, the identification of vimentin (vimentin354), a nuclear component in GBM cells, as the main target of sumoylation promoted by PIAS3.

Matrix-assisted laser desorption/ionization time-of-flight analysis of low-concentration oligonucleotides and mini-sequencing products.

A new sample preparation procedure has been developed to improve the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of low-concentration oligonucleotides. In this method, a few microL of the oligonucleotide solutions are first dispensed and allow drying and shrinking to a small spot on an anchoring target. Thereafter, a small volume (0.1 microL) of the saturated 3-hydroxypicolinic acid (3-HPA) solution is added to the top of the shrunk oligonucleotide spot. It is found that the sample prepared by this procedure is more homogenous and substantially reduces the need to search for 'sweet' spots. Importantly, the increased shot-to-shot and sample-to-sample reproducibility makes it possible to perform high-throughput and quantitative analysis. We demonstrated that this procedure allowed the robust detection of oligonucleotides at a level of 0.01 microM and mini-sequencing products produced using only 50 fmol of the extension primer.

Capillary electrochromatographic separation of enantiomers on chemically bonded type of cellulose derivative chiral stationary phases with a positively charged spacer.

Positively charged chiral stationary phases (CSPs) were prepared for capillary electrochromatography (CEC) separation of enantiomers by chemically immobilizing cellulose derivatives onto diethylenetriaminopropylated silica (DEAPS) with tolylene-2,4-diisocyanate (TDI) as a spacer reagent. Anodic electroosmotic mobility was observed in both nonaqueous and aqueous mobile phases due to the positively charged amines on the surface of the prepared CSPs. For comparison, the traditionally used 3-aminopropyl silica (APS) was also adopted as the base material instead of DEAPS to prepare CSP. It was observed that the EOF on the DEAPS-based CSP was 18%-60% higher than that on the APS-based CSP under nonaqueous mobile phase conditions. Separation of enantiomers in CEC was performed on the positively charged CSPs with the nonaqueous mobile phases of pure ethanol or mixture of hexane-alcohol and the aqueous phases of acetonitrile-water or 95% ethanol. Fast separation of enantiomers was achieved on the newly prepared CSPs.

A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry.

We report a novel method termed matrix suppressed laser desorption/ionization to improve the analysis of low-mass molecules by MALDI-TOF mass spectrometry. In this method, the surfactant of cetrimonium bromide (CTAB) is added to the conventional matrix of alpha-cyano-4-hydroxycinnamic acid solution to prepare the MALDI samples. During the MALDI process, the presence of CTAB could substantially or even completely suppress the matrix-related ion background. As a result, very clean mass spectra can be routinely obtained in the low-mass range. In addition, the presence of CTAB can significantly improve the mass resolution of low-mass molecules. It is seen that high-quality spectra were routinely obtained at a matrix/CTAB ratio of 1000:1. This method has been successfully used to analyze a variety of low-mass molecules.

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry.

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH(4)HF(2). The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50 degrees C, even 20 fmol cytochrome c could be well digested and detected.

Carbon nanotubes as assisted matrix for laser desorption/ionization time-of-flight mass spectrometry.

Analysis of low molecular weight compounds with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed by using carbon nanotubes obtained from coal by arc discharge as the matrix. The carbon nanotube matrix functions as substrate to trap analytes of peptides, organic compounds, and beta-cyclodextrin deposited on its surface. It has been found that carbon nanotubes can transfer energy to the analyte under laser irradiation, which makes analytes well desorbed/ionized, and the interference of intrinsic matrix ions can be eliminated. At the same time, the fragmentation of the analyte can be avoided. A good sensitivity and excellent reproducibility of the spectrum signals are achieved. It is believed that this work not only will open a new field for applications of carbon nanotubes, but also will offer a new technique for high-speed analysis of low molecular weight compounds in areas such as metabolism research and characterization of natural products.

Unilamellar liposomes covalently coupled on silica gel for liquid chromatography.

Silica gel was used as a support for the covalent coupling of liposomes, which could overcome drawbacks of soft gel beads in column efficiency and separation speed. The influences of the concentration of added dimethylaminopyridine and reaction time on the chloroformate activation reaction of silica gel were investigated. Temperature and pH for covalent coupling of liposomes on the activated silica gel were also optimized. Experimental results indicated that the stability of the covalently coupled liposome columns was obviously superior to that of the noncovalently coated liposome columns but the selectivity of both columns was basically identical. Separation and analysis of a crude extract of a traditional Chinese medicine Ligusticum Wallichii and a mixture of small peptides on both columns further support this conclusion.