Structural and functional biomimetics of [Fe]-hydrogenase featuring a mono-, di- or tetrasubstituted pyridine ligand with a fac-C, N, and S ligation.

The combined structural and functional modeling study of [Fe]-H2ase has remained a great challenge, to date. Now, we report a series of new structural and functional [Fe]-H2ase models (1-6) that contain a mono-, di- or tetrasubstituted pyridine ligand with a biomimetic fac-C, N, and S ligation. While models 1-3, 5 and 6 are conveniently prepared by a novel flexible pyridine ligand (FPL)-based method, model 4 is prepared simply by an intramolecular CO replacement reaction of model 3. More interestingly, the structural study by spectroscopy and X-ray crystallography proves that these new models include a biomimetic fac-acyl (or methylene) C, pyridyl N, and thioether S ligation to an Fe(II) center and the C-Fe(II) σ bond is trans to an iodo ligand. In addition, the chemical reactivity study proves that they all have the enzyme-like H2 activation and hydride transfer functions in the presence of imidazolium Im+, AgBF4 and Et3N. Particularly interesting is that a possible pathway for such H2 activation and hydride transfer reactions catalyzed by a representative model 4 is proposed and the existence of the highly unstable 5-coordinate intermediate M4 and Fe-H species M5 is supported by the isolation and characterization of their MeCN-coordinated derivative 7 and chloro-substituted derivative 8, respectively.

Biomimetic models of [Fe]-hydrogenase featuring a 2-acylphenylthiomethyl-6-R-pyridine (R = H or OMe) ligand.

Despite a variety of [Fe]-H2ase models prepared so far, the structural and functional modeling study of the enzyme has remained a great challenge. Now, we report a new type of flexible pyridine ligand (FPL)-based synthetic method by which two novel [Fe]-H2ase models have been prepared. Notably, the two models contain not only a biomimetic fac-acyl C, pyridyl N, thioether S coordination mode but also possess the enzyme-like H2/D2 activation functions.

Weighted Gene Coexpression Network Analysis Reveals Essential Genes and Pathways in Bipolar Disorder.

Bipolar disorder (BD) is a major and highly heritable mental illness with severe psychosocial impairment, but its etiology and pathogenesis remains unclear. This study aimed to identify the essential pathways and genes involved in BD using weighted gene coexpression network analysis (WGCNA), a bioinformatic method studying the relationships between genes and phenotypes. Using two available BD gene expression datasets (GSE5388, GSE5389), we constructed a gene coexpression network and identified modules related to BD. The analyses of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways were performed to explore functional enrichment of the candidate modules. A protein-protein interaction (PPI) network was further constructed to identify the potential hub genes. Ten coexpression modules were identified from the top 5,000 genes in 77 samples and three modules were significantly associated with BD, which were involved in several biological processes (e.g., the actin filament-based process) and pathways (e.g., MAPK signaling). Four genes (NOTCH1, POMC, NGF, and DRD2) were identified as candidate hub genes by PPI analysis and CytoHubba. Finally, we carried out validation analyses in a separate dataset, GSE12649, and verified NOTCH1 as a hub gene and the involvement of several biological processes such as actin filament-based process and axon development. Taken together, our findings revealed several candidate pathways and genes (NOTCH1) in the pathogenesis of BD and call for further investigation for their potential research values in BD diagnosis and treatment.

Forensic and phylogenetic analyses of populations in the Tibetan-Yi corridor using 41 Y-STRs.

Y-chromosome haplotypes of 527 non-related males (176 Han, 186 Tibetan, and 165 Yi) in the Tibetan-Yi corridor were analyzed using SureID® PathFinder Plus. In the populations of Han, Tibetans, and Yi, the haplotype diversity was 0.9989, 0.9981, and 0.9993, respectively, and the discrimination capacity was 0.9148, 0.8925, and 0.9576, respectively. Phylogenetic relationships among 12 studied ethnic groups and 7 other ethnic groups in the Tibetan-Yi corridor were investigated. Both multi-dimensional scaling analysis and phylogenetic reconstructions indicated that Tibetans appeared separated from the Han and Yi ethnic groups in the Tibetan-Yi corridor. Their genetic homogeneity or heterogeneity has not entirely been affected by their geographical distance and linguistic origin.

Purification, structure characterization and antioxidant activity of polysaccharides from Saposhnikovia divaricata.

Polysaccharide from traditional Chinese herb, Saposhnikovia divaricata (Turcz.) Schischk. (SD) was extracted, fractionated and characterized in this work. Four fractions were prepared. Their molecular weight, monosaccharide compositions, linkage modes and structural properties were characterized with SEC-MALS-RI, HPAEC-PAD, GC-MS and NMR. SDP1 was assigned as a 1, 4-α-glucan with small amount of O-6 linked branches. SDP2 contained a big amount of the 1, 4-α-glucan and a small amount of arabinogalactan, while SDP3 possessed relatively lower amount of the 1, 4-α-glucan and a big amount of the arabinogalactan. SDP4 was defined as a pectic arabinogalactan. Four fractions showed antioxidant activities in both molecular and cellular levels and their activity was ranked as SDP4 ≈ SDP3>SDP2>SDP1. The 1, 4-α-glucan in SDP1 had the weakest, while SDP3 and SDP4 showed similar and the highest antioxidant activity. The arabinogalactan was the major component of both SDP3 and SDP4, which significantly contributed to the antioxidant activity of SDP.

Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis.

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis. In this study, we purified a novel protease inhibitor (bdellin-HM-2) with anticoagulant properties from H. manillensis. With a molecular weight of 1.4x104, bdellin-HM-2 was also characterized with three intra-molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDD motifs at the C-terminus. cDNA cloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 amino acids and was encoded by a 399 bp open reading frame (ORF). Sequence alignment showed that bdellin-HM-2 shared similarity with the "non-classical" Kazal-type serine protease inhibitors, but had no inhibitory effect on trypsin, elastase, chymotrypsin, kallikrein, factor XIIa (FXIIa), factor XIa (FXIa), factor Xa (FXa), thrombin, or plasmin. Bdellin-HM-2 showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT), indicating a role in enabling H. manillensis to obtain a blood meal from its host. Our results suggest that bdellin-HM-2 may play a crucial role in blood-sucking in this leech species and may be a potential candidate for the development of clinical anti-thrombotic drugs.

In vitro and in vivo evaluation of hypothermia on pharmacokinetics and pharmacodynamics of nimodipine in rabbits.

Objective To investigate the effect of hypothermia on the pharmacokinetics and pharmacodynamics of nimodipine in rabbits using in vivo and in vitro methods. Methods Five healthy New Zealand rabbits received a single dose of nimodipine (0.5 mg/kg) intravenously under normothermic and hypothermic conditions. Doppler ultrasound was used to monitor cerebral blood flow, vascular resistance, and heart rate. In vitro evaluations of protein binding, hepatocyte uptake and intrinsic clearance of liver microsomes at different temperatures were also conducted. Results Plasma concentrations of nimodipine were significantly higher in hypothermia than in normothermia. Nimodipine improved cerebral blood flow under both conditions, but had a longer effective duration during the hypothermic period. Low temperature decreased the intrinsic clearance of liver microsomes, with no change in protein binding or hepatocyte uptake of nimodipine. Conclusion Nimodipine is eliminated at a slower rate during hypothermia than during normothermia, mainly due to the decreased activity of cytochrome P450 enzymes. This results in elevated system exposure with little enhancement in pharmacological effect.

Auto-Induction Effect of Chloroxoquinoline on the Cytochrome P450 Enzymes of Rats Associated with CYP 3A and 1A.

To investigate the auto-induction of cytochrome P450 (CYP450) by Chloroxoquinoline (CXL), a novel anticancer drug. Three experiments related to the induction of CYP450 were performed: a) In vitro use of the rat fresh hepatocytes model; b) In vivo 'cocktail' of CYP450 probe model; c) Pharmacokinetic (PK) study of the single and multiple doses. Some typical CYP enzyme probes and inducers were used in these experiments and were all determined by HPLC-MS/MS. The expression levels of CYP3A and CYP1A mRNA were analyzed by the real time polymerase chain reaction (RT-PCR) technique. The PK studies showed that the area under the curve (AUC0-t) and the peak concentration (Cmax) of the multiple doses were approximately 2.4-fold and 1.9-fold lower than those of the single dose, respectively (p < 0.05). Subsequent studies were conducted to study the possible induction of CXL on CYP 450. The in vivo 'cocktail' administration of CYP450 probe model indicated that 5 d pretreatment with CXL resulted in a mean 4.6 times increase in the metabolites/probe plasma ratios for CYP 3A and a 336% increase for CYP 1A than those of the negative control (p < 0.05). The induction effect of CXL on CYP450 was further evaluated on rat hepatocytes with four concentrations (1, 10, 50 and 100 µmol/L). Compared with the negative control, the mRNA levels of CYP 1A2 increased significantly in rat hepatocytes after treatment with 10, 50 and 100 µmol/L CXL (p < 0.05). While significant inductions of CYP 3A1 were observed in the entire treated groups. The results of the present study demonstrate enhanced and induced expression of CYP 3A and CYP 1A in response to CXL exposure in rats, suggesting that CXL is an auto-inducer of CYP 3A and CYP 1A.

In vitro evaluation of 9-(2-phosphonylmethoxyethyl)adenine ester analogues, a series of anti-HBV structures with improved plasma stability and liver release.

Chronic hepatitis B virus (HBV) infection may lead to liver cirrhosis and hepatocellular carcinoma, but few drugs are available for its treatment. Acyclic nucleoside phosphonates (ANPs) have remarkable antivirus activities but are not easily absorbed from the gastrointestinal tract and accumulate in the kidneys, resulting in nephrotoxicity. Therefore, there is a need to find effective liver site-specific prodrugs. The dipivaloyloxymethyl ester of 9-(2-phosphonylmethoxyethyl)adenine (PMEA)-adefovir dipivoxil (ADV)-is a first-line therapy drug for chronic hepatitis B with a low therapeutic index because of renal toxicity and low hepatic uptake. In this study, a series of PMEA derivatives were synthesized to enhance plasma stability and liver release. The metabolic stability of ADV (Chemical I) and its two analogues (Chemicals II and III) was evaluated in rat plasma and liver homogenate in vitro. An ion-pair reverse-phase HPLC-UV method and a hybrid ion trap and high-resolution time-of-flight mass spectrometry (LC-IT-TOF-MS) were used to evaluate the degradation rate of the analogues and to identify their intermediate metabolites, respectively. Chemicals I and II were hydrolyzed by cleavage of the C-O bond to give monoesters. Sufficient enzymatic activation in the liver homogenate through a relatively simple metabolic pathway, in addition to a favorable stability profile in rat plasma, made Chemical II an optimal candidate. Next, six analogues based on the structure of Chemical II were synthesized and evaluated in plasma and liver homogenate. Compared to Chemical II, these compounds generated less active PMEA levels in rat liver homogenate. Therefore, chemical modification of Chemical II may lead to new promising PMEA derivatives with enhanced plasma stability and liver activation.

Simultaneous determination of triptolide and its prodrug MC002 in dog blood by LC-MS/MS and its application in pharmacokinetic studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii HOOK F (TWHF) is a traditional Chinese medicine used in the treatment of various autoimmune diseases and inflammatory disorders including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and skin diseases. Triptolide (TP) is one of the main active ingredients of this traditional Chinese medicine. MC002 is a novel semi-synthetic derivate of TP which is highly water soluble, acts as a prodrug and is converted to TP in vivo. AIM OF THIS STUDY: A sensitive, rapid method for the simultaneous determination of TP and its chemo-unstable prodrug MC002 in dog blood was developed and validated using electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this method, a pharmacokinetic study of MC002 and TP following an intravenous drip infusion of 0.2mg/kg MC002 in dogs was performed. MATERIALS AND METHODS: Chemo-degradation of the prodrug in blood samples was inhibited by the addition of a small amount of sodium fluoride solution before using liquid-liquid extraction with ethyl acetate. The concentrations of MC002 and TP in dog blood were determined using the LC-MS/MS method. RESULTS: The quantitative method showed good precision and stability and is suitable for the assay of biological samples. The pharmacokinetic study showed that the elimination of MC002 was faster than that of TP, and the concentrations and AUC0-t values of TP were higher than MC002. MC002 can rapidly convert to TP in vivo. CONCLUSIONS: This validated method was successfully applied in a pharmacokinetic study of MC002 following an intravenous drip infusion in dogs. With the development of this new prodrug of TP as a promising anti-cancer drug, this method is suitable for its further analysis in clinical studies.

Rapid screening of drug-protein binding using high-performance affinity chromatography with columns containing immobilized human serum albumin.

For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 - 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation.

[Determination of morroniside concentration in rat plasma by high performance liquid chromatography-tandem mass spectrometry].

Morroniside, an iridoid glycoside extracted from Cornus officinalis, has multiple pharmacological effects such as neuroprotection. This study took the lead in establishing a method for determining morroniside concentration in rat plasma by high performance liquid chromatography-tandem mass spectrometry. Plasma samples were processed with protein precipitation method, with hyperoside as the internal standard. An Inertsil C8-3 column (2. 1 mm x 50 mm, 5 microm) was adopted, with a mobile phase composed of water (containing 1 mmol L-1 Sodium formate)-acetonitrile (gradient elution) at a flow rate of 0.4 mL . min -1. Electrospray ionization (ESI) was adopted in the positive ion mode for multi-reaction monitoring (MRM). Morroniside showed a good linear relationship ranging between 2-5 000 microg L-1 (r = 0. 995 7), with the minimum limit of quantification of 2 microg L-1. Its precise, accuracy, recovery and matrix effect were all in line with the biological sample measurement requirements. Therefore, the method described above was proved to be suitable for the determination of morroniside concentration in rat plasma. To use the method in the pharmacokinetic study on morroniside in rats, oral administration dose shall be set at 20 mg . kg - to map the plasma concentration-time curve. Main pharmacokinetic parameters were calculated by DAS 2. 0. Specifically, AUC0-inifinity was (587.6 +/- 290. 7) microg min L-1, Cmax was (334.2+/-148.0) microg L-1, Tmax was (0.6 +/-0.3) h, t1/2 was (0.7+/-0.3) h.

[Research progress of enhancing quantitative sensitivity by using LC-MS(n) with derivatization method in bio-matrices].

Liquid chromatography/mass spectrometry (LC-MS(n)) has been essential to a large number of quantitative analytical applications in drug research, and especially in the drug PK/PD research, due to its high sensitivity and high specificity. But following the appearance of drugs with high activity and low dosage and the especial structural compounds, a number of limitations of LC-MS(n) have been noted. Derivatization changes the structure of drugs and therefore changes their physical and chemical properties, resulting in high ionization efficiency, low matrix effect and low disturbance by inorganic salts and endogenous compounds in LC-MS(n). In this article, recent progress in the research of the chemical derivatization strategy with LC-MS(n) is reviewed on breakthrough of some LC-MS(n) limitations, in particular focusing on the applications involving some drugs in bio-matrices.

Plasma membrane as the target site of cholic acid analogs.

Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in cellular membrane fluidity was supported by: ileal contraction that was induced by acetylcholine and mediated by the muscarinic receptor (M-receptor) or the relaxation induced by adrenaline and mediated by the ß-adrenergic receptor (ß-receptor) was inhibited by CAAs. They also had similar rank-order of potency and the effects on the plasma membrane. Collectively, the plasma membrane may be a target for the CAAs to exert the multiple pharmacological effects which are mediated by the alteration of the membrane mobility and the function of integral membrane proteins.

Pharmacokinetic properties of albiflorin and paeoniflorin after oral administration of pure compound, Radix Paeoniae alba extract and danggui-shaoyao-san extract to rats.

This study compared the pharmacokinetics of albiflorin (ALB) and paeoniflorin (PAE), respectively, after oral administration of ALB, PAE, Radix Paeoniae alba (RPA) extract, and Danggui-Shaoyao-San (DSS) extract to rats on separate occasions. Analytes were detected simultaneously with liquid chromatography-tandem mass spectrometry. Noncompartmental pharmacokinetic parameters were calculated. After oral administration of RPA and DSS extract to rats, ALB reached maximum concentrations of 4637 ± 2774 ng/ml (0.40 ± 0.14 h) and 226 ± 122 ng/ml (0.35 ± 0.14 h) and PAE reached maximum concentrations of 2132 ± 560 ng/ml (0.40 ± 0.14 h) and 143 ± 65 ng/ml (0.45 ± 0.11 h), respectively. Compared to the AUC(0 - t) value (1122 ± 351 and 722 ± 158 ng h/ml for ALB and PAE, respectively) after administration of monomers, larger AUC(0 - t) value of ALB (4755 ± 2560 ng h/ml) and PAE (2259 ± 910 ng h/ml) after administration of RPA extract and smaller AUC(0 - t) value of ALB (411 ± 118 ng h/ml) and PAE (242 ± 126 ng h/ml) after administration of DSS extract were obtained. The C(max), AUC, and K(el) of ALB and PAE were remarkably increased (P < 0.05, 0.01 or 0.005) during oral administration of RPA extract in comparison to that of DSS extract.

Liquid chromatography-tandem mass spectrometry for the determination of paclitaxel in rat plasma after intravenous administration of poly(L-glutamic acid)-alanine-paclitaxel conjugate.

A specific and sensitive liquid chromatography-tandem mass spectrometric method for quantitative determination of paclitaxel in rat plasma was developed and validated using docetaxel as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma. The separation of paclitaxel was performed on a C(18) column with a mobile phase of acetonitrile:water:formic acid (65:35:0.1, v/v/v) over 5min. The assay was based on the selected reaction monitoring transitions at m/z of the precursor-product ion transitions m/z 854.2-->286.1 for paclitaxel and 808.3-->527.2 for internal standard. The lower limit of quantification was 0.5ng/mL based on 100microL of plasma. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 95.4 and 105.4%. The extraction recoveries ranged from 96.7 to 103.7% across the calibration curve range. The method was successfully applied to measurement of low concentrations of paclitaxel or regenerated paclitaxel in plasma after intravenous administration of a single dose (10mg/kg) of a poly(l-glutamic acid)-alanine-paclitaxel conjugate to rats.

Metabolic stability of human parathyroid hormone peptide hPTH (1-34) in rat tissue homogenates: kinetics and products of proteolytic degradation.

The present study aim to investigate the metabolic stability and degradation of cleavage sites of human parathyroid hormone peptide, hPTH (1-34), in rat tissue homogenate, and to identify the types of proteases involved in hPTH (1-34) processing degradation. The stability of hPTH (1-34) in rat kidney, lung and liver homogenates was evaluated by LC-ESI-MS, and the structures of the major degradation products were identified by MALDI-TOF MS and LC-ESI-MS/MS. The ability of protease inhibitors to inhibit hPTH (1-34) degradation was used to identify the class of proteases involved in the metabolism of hPTH (1-34). hPTH (1-34) peptide was readily degraded in rat kidney, liver, and lung homogenates, with half-lives of 5.7, 32.2, and 18.9 min, respectively. The degradation of hPTH (1-34) in each tissue can be inhibited by inhibitors of serine and metalloproteases. The major degradation products of hPTH (1-34) are similar in each tissue and suggest that hPTH (1-15) and hPTH (16-34) appear as the major degradation products. The degradation patterns of hPTH (1-34) incubated in rat kidney, liver and lung homogenates are largely overlapping, and a majority of the fragments are generated via cleavages at sites of Leu15-Asn16 peptide bond.

[Determination of decapeptide LXT-101 in plasma by HPLC-MS/MS and its pharmacokinetics in Beagle dogs].

This paper developed a sensitive and specific liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/MS) method for the determination of decapeptide LXT-101 in Beagle dog plasma. Plasma samples spiked with internal standard (IS) were treated with acetonitrile to precipitate the protein. Selected reaction monitoring (SRM) using the precursor --> product ion combinations of m/z 472.1-->587.9 and m/z 502.8-->633.8 were used to quantify LXT-101 and IS, respectively. The linear calibration curves were obtained in the concentration range of 0.5 - 500.0 ng x mL(-1). The limit of quantification (LOQ) was 0.5 ng x mL(-1). The inter-day and intra-day precision (RSD) across three validation run over the entire concentration range was below 10.9%, and the accuracy (RE) was within +/- 1.8%. The main pharmacokinetic parameters of LXT-101 after muscle injection of 20 microg x kg(-1) were as follows, AUC(0-t): (176.8 +/- 116.7) microg x h x L(-1), MRT(0-t): (2.52 +/- 0.53) h, T(1/2): (1.4 +/- 0.3) h; CL: (0.16 +/- 0.09) L x h(-1) x kg(-1), and Vd: (0.30 +/- 0.16) L x kg(-1), respectively. The method is proved to be specific, sensitive and suitable for the investigation of LXT-101 pharmacokinetics in Beagle dog.

Degradation of salmon calcitonin in rat kidney and liver homogenates.

Salmon calcitonin (sCT), a 32-amino-acid peptide, is the active component in many pharmaceuticals used for the management of bone diseases. In this study, the stability of sCT in rat kidney and liver homogenates were evaluated by LC-ESI-MS, and the structures of the major degradation products were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results show that the half life of sCT was 13.18 min in rat kidney homogenate (2.5 mg/ml, protein concentration) and 43.07 min in rat liver homogenate (2.5 mg/ml, protein concentration). MALDI-TOF MS results indicated that sCT was initially cleaved at Leu9-Gly10 and Gly10-Lys11 bonds in rat kidney homogenate in vitro, at the same time, the major degradation fragment, Lys11-Pro32-NH2 Was metabolized at the C-terminal amide by deamidation, whereas in rat liver homogenate, the initial cleavage sites were at Val8-Leu9 and His17-Lys18. The results indicated that the metabolism of sCT proceeds by initial endoproteolytic cleavage and subsequent exoproteolytic digestion.

Determination of 4-dimethylaminophenol concentrations in dog blood using LC-ESI/MS/MS combined with precolumn derivatization.

A sensitive and reproducible LC-ESI/MS/MS method, which was combined with the precolumn dansyl chloride derivatization to enhance the signal intensity of analytes, was developed to determine blood 4-dimethylaminophenol (DMAP) concentrations. The linearity of the method was observed within the concentration range of 2-2000 ng/mL. The precision, accuracy, stability, recovery and matrix effect of the method were also investigated and found to meet the requirements for pharmacokinetic studies of the drug. By using this method, pharmacokinetic studies were conducted in dogs after i.m. and i.v. administrations. The results showed that DMAP could not only be absorbed into blood quickly after i.m., but also can be eliminated rapidly. Both i.m. and i.v. routes are appropriate for DMAP to be used in field first-aid. It has been proved that this LC-MS/MS combined with precolumn derivatization method can be used as a routine analytical method to provide enhanced measurements for blood DMAP concentrations. It is also useful for DMAP pharmacokinetic evaluation.