Transcriptomic Analysis Reveals That Granulocyte Colony-Stimulating Factor Trigger a Novel Signaling Pathway (TAF9-P53-TRIAP1-CASP3) to Protect Retinal Ganglion Cells after Ischemic Optic Neuropathy.

Optic nerve head (ONH) infarct can result in progressive retinal ganglion cell (RGC) death. The granulocyte colony-stimulating factor (GCSF) protects the RGC after ON infarct. However, protective mechanisms of the GCSF after ONH infarct are complex and remain unclear. To investigate the complex mechanisms involved, the transcriptome profiles of the GCSF-treated retinas were examined using microarray technology. The retinal mRNA samples on days 3 and 7 post rat anterior ischemic optic neuropathy (rAION) were analyzed by microarray and bioinformatics analyses. GCSF treatment influenced 3101 genes and 3332 genes on days 3 and 7 post rAION, respectively. ONH infarct led to changes in 702 and 179 genes on days 3 and 7 post rAION, respectively. After cluster analysis, the levels of TATA box-binding protein (TBP)-associated factor were significantly reduced after ONH infarct, but these significantly increased after GCSF treatment. The network analysis revealed that TBP associated factor 9 (TAF9) can bind to P53 to induce TP53-regulated inhibitor of apoptosis 1 (TRIAP1) expression. To evaluate the function of TAF9 in RGC apoptosis, GCSF plus TAF9 siRNA-treated rats were evaluated using retrograde labeling with FluoroGold assay, TUNEL assay, and Western blotting in an rAION model. The RGC densities in the GCSF plus TAF9 siRNA-treated rAION group were 1.95-fold (central retina) and 1.75-fold (midperipheral retina) lower than that in the GCSF-treated rAION group (p < 0.05). The number of apoptotic RGC in the GCSF plus TAF9 siRNA-treated group was threefold higher than that in the GCSF-treated group (p < 0.05). Treatment with TAF9 siRNA significantly reduced GCSF-induced TP53 and TRIAP1 expression by 2.4-fold and 4.7-fold, respectively, in the rAION model. Overexpression of TAF9 significantly reduced apoptotic RGC and CASP3 levels, and induced TP53 and TRIAP1 expression in the rAION model. Therefore, we have demonstrated that GCSF modulated a new pathway, TAF9-P53-TRIAP1-CASP3, to control RGC death and survival after ON infarct.

Simultaneous determination of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry.

The detection and confirmation of cannabinoids in oral fluid are important in forensic toxicology. Currently, the presence of Δ(9)-tetrahydrocannabinol (THC) is used for the detection of cannabis in oral fluid. A low concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) is found in oral fluid, which suggested a convenient and low-sensitivity confirmation assay can be used in a routine forensic laboratory. In this study, a highly sensitive isotope dilution liquid chromatography-tandem mass spectrometry method following dansylation was successfully developed for simultaneous determination of THC and THC-COOH in oral fluid. The dansylated derivatives dramatically demonstrated and enhanced the sensitivity of THC and THC-COOH. To avoid signal influenced by the matrix, a 5-min liquid chromatography gradient program was evaluated and optimized, which reduced the sample diffusion and caused sharp peaks (less than 12 s) and thus helped to achieve detection at a low level. The sensitivity, accuracy, and precision were also evaluated, and high quantitative accuracy and precision were obtained. The limit of quantitation of this approach was 25 pg/mL for THC and 10 pg/mL for THC-COOH in oral fluid. Finally, the method was successfully applied to eight suspected cannabis users. Among them, in six oral fluid samples THC-COOH was determined at a concentration from 13.1 to 47.2 pg/mL.

Combined Dynamic Alterations in Urinary VEGF Levels and Tissue ADAM9 Expression as Markers for Lethal Phenotypic Progression of Prostate Cancer.

Recent evidence has demonstrated that detection of changes in the levels of urinary vascular endothelial growth factor (VEGF) and tissue a disintegrin and metalloproteinase 9 (ADAM9) is effective in determining prostate cancer progression. To evaluate the combined application of VEGF and ADAM9 as early progression markers of lethal phenotypic cancer, quantification of urinary VEGF and tissue ADAM9 expression was studied in patients with late stage prostate cancer. Tissue biopsies were collected during palliative transurethral resection of prostate (TURP) surgery, and urine samples were collected before hormone therapy and 3, 6 and 12 months post-TURP. We observed a nearly 100% correlation between increasing urinary VEGF levels over time and prostate cancer progression, but no correlation was observed when comparing urinary VEGF concentrations at a single time point and cancer progression. In addition, we also observed correlation of increasing ADAM9 nuclear positive staining and lethal phenotypic transition. Statistical analysis revealed that both the increase in urinary VEGF level and the presence of the tissue ADAM9 nuclear staining were significantly correlated with the risk of patients with relapse prostate cancer (P < 0.05). Thus, we suggest that combination of detection of changes in urinary VEGF and tissue staining of ADAM9 may be accurate for predicting the mortality of patients with prostate cancer during hormone therapy.

Long term therapeutic effects of icariin-loaded PLGA microspheres in an experimental model of optic nerve ischemia via modulation of CEBP-ß/G-CSF/noncanonical NF-κB axis.

An ischemic insult at optic nerve (ON) is followed by detrimental neuroinflammation that results in progressive and long-lasting retinal ganglion cell (RGC) death and vision loss. Icariin was reported to be a safe and effective natural anti-inflammatory drug. Herein, we evaluated the long-term therapeutic effects of a single intravitreal injection of poly(lactide-co-glycolide) PLGA-icariin in a rat model of anterior ischemic optic neuropathy (rAION). Treatment with PLGA microspheres of icariin preserved the visual function and RGC density for 1 month in the rAION model. In addition, ON edema and macrophage infiltration were inhibited by treating PLGA microspheres of icariin. We found that the binding complex of icariin and CCAAT enhancer binding protein beta (CEBP-ß) significantly induced endogenous granulocyte colony-stimulating factor (G-CSF) expression to activate noncanonical nuclear factor kappa B (NF-κB) signaling pathway by promoting an alternative phosphorylation reaction of IKK-ß. Activation of noncanonical NF-κB signaling pathway promoted the M2 microglia/macrophage polarization and AKT1 activation, which prevented neuroinflammation and RGC apoptosis after ON infarct. This study concluded that protective mechanism of icariin is a CEBP-ß/G-CSF axis-induced noncanonical NF-κB activation, which provides the long-term neuroprotective effects via anti-inflammatory and antiapoptotic actions after ON ischemia.

Isoamylamine Induces B16-F1 Melanoma Cell Autophagy by Upregulating the 5' Adenosine Monophosphate-Activated Protein Pathway.

Isoamylamine (IA) is an aliphatic monoamine molecule present in cheese, eggs, and wine. It belongs to the family of polyamines and also can be synthesized endogenously. It has been known that regulation of polyamines in cells is related to cell cycle and tumor formation. Malignant melanoma is difficult to treat and easily resistant to chemotherapy/radiotherapy through autophagy. In this study, we aim to clarify whether IA has a growth control effect on melanoma tumor cells and the regulatory mechanism. We treated B16-F1 melanoma cells with IA at concentrations of 0, 200, 400, and 600 ppm for 24 h. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was checked for cell viability and results showed that IA has an inhibitory effect on B16-F1 melanoma cells. The signaling molecules, which included Raf/MEK/ERK, were activated, while MSK1 and protein kinase B (AKT) were suppressed. Autophagy was also confirmed to be induced by IA. The acridine orange stain-positive cells were increased and BECN-1/LC3 upregulated. The data also showed that the autophagy regulatory molecule, 5'-adenosine monophosphate-activated protein kinase (AMPK), was induced after IA treatment, so we used dorsomorphin to inhibit AMPK and found that it could suppress autophagy. In conclusion, IA has an effect of inducing autophagy in B16-F1 cells and it is regulated through AMPK.

Inhibition of Retinal Ganglion Cell Loss By a Novel ROCK Inhibitor (E212) in Ischemic Optic Nerve Injury Via Antioxidative and Anti-Inflammatory Actions.

Purpose: This study investigated the neuroprotective effects of administration of ROCK inhibitor E212 on ischemic optic neuropathy. Methods: Rats received an intravitreal injection of either E212 or PBS immediately after optic nerve infarct. The oxidative stress in the retina was detected by performing superoxide dismutase activity and CellROX assays. The integrity of retinal pigment epithelium was determined by staining of zona occludens 1. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined by using flash visual-evoked potential analysis, retrograde FluoroGold labeling, and TdT-dUTP nick end-labeling assay. Macrophage infiltration was detected by staining for ED1. The protein levels of TNF-α, p-CRMP, p-AKT1, p-STAT3, and CD206 were evaluated using Western blotting. Results: Administration of E212 resulted in a 1.23-fold increase in the superoxide dismutase activity of the retina and 2.28-fold decrease in RGC-produced reactive oxygen species as compared to the levels observed upon treatment with PBS (P < 0.05). Moreover, E212 prevented the disruption of the blood-retinal barrier (BRB) in contrast to PBS. The P1-N2 amplitude and RGC density in the E212-treated group were 1.75- and 2.05-fold higher, respectively, than those in the PBS-treated group (P < 0.05). The numbers of apoptotic RGCs and macrophages were reduced by 2.93- and 2.54-fold, respectively, in the E212-treated group compared with those in the PBS-treated group (P < 0.05). The levels of p-AKT1, p-STAT3, and CD206 were increased, whereas those of p-PTEN, p-CRMP2, and TNF-α were decreased after treatment with E212 (P < 0.05). Conclusions: Treatment with E212 suppresses oxidative stress, BRB disruption, and neuroinflammation to protect the visual function in ischemic optic neuropathy.

Integration of GC/EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair.

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography-mass spectrometry (GC-MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol-trifluoroacetic acid (methanol-TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives analyzed by GC-MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.

Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction.

This study describes the development of a simple RT-PCR method to amplify the whole genome of the influenza A virus based on the amplification of full-length gene segments. Primers were designed based on the conserved regions of both the 5'-end and the 3'-end of each gene segment. After optimizing the duration and temperature of denaturing, annealing, and extension, these primers could amplify all of the full-length gene segments. To test the accuracy of the method, all amplicons were subjected to DNA sequencing with an autosequencer. Eighteen strains of influenza A virus which belonged to H1N1 or H3N2 subtypes were tested. All eight segments of both subtypes were successfully amplified in all tested strains. Using a newly developed reverse-transcriptase (RT), primers and PCR running conditions, this study established a protocol to amplify the entire genome of the influenza A virus. This method provides a tool which can be used for the amplification of all genes of the H1N1 and H3N2 subtypes of influenza A virus prior to analysis of their sequences, and to construct expression plasmids for further study.

Induction of caspase-3-dependent apoptosis in human leukemia HL-60 cells by paclitaxel.

BACKGROUND: Paclitaxel, an antineoplastic drug, inhibits cell growth and cell cycle progression and induces apoptosis in human leukemia HL-60 cells. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. METHODS: We examined the cell morphology and apoptosis in HL-60 cells after exposure to paclitaxel and measured caspase-3 activities with or without z-VAD-fmk (a broad-spectrum caspase inhibitor) pretreatment by flow cytometric analysis and Western blotting. RESULTS: Together, our results were (1) paclitaxel mainly induced G2/M cell cycle arrest in HL-60 cells (p<0.001); (2) time (p<0.001)- and dose-dependent (p<0.001) apoptosis of HL-60 cells was induced by paclitaxel; (3) in HL-60 cells, z-VAD-fmk blocked paclitaxel-induced apoptosis (12 h: p<0.001; 24 h: p<0.01; 48 h: p<0.01; 72 h: p<0.001) and caspase-3 activation (12 h: p<0.05; 24 h: p<0.01; 48 h: p<0.01; 72 h: p<0.01). CONCLUSIONS: These results suggest that paclitaxel can induce G2/M cell cycle transition and apoptosis via caspase-3 activity in HL-60 cells.

The correlation between CpG methylation on promoter and protein expression of E-cadherin in oral squamous cell carcinoma.

BACKGROUND: Reduction of E-cadherin in most common epithelial tumors relates to metastasis, which results from the silence of E-cadherin by CpG methylation. MATERIALS AND METHODS: We examined the E-cadherin expression by immunohistochemical staining and detected methylation by methylation-specific polymerase chain reaction (MSP) in 48 primary oral SCC tissues. RESULTS: The results showed that 41 out of 48 (85.4%) cancerous tissues and 16 out of 48 (33.3%) nearby non-cancerous tissues had CpG methylation on the promoter region of E-cadherin. In these non-cancerous tissues, 2 out of 16 (12.5%) had no methylation change in their paired cancerous part. Immunohistochemical study showed that a decreased expression pattern was found in the tissue which had CpG methylation on the promoter region, but an over expression island or aberrant expression was also frequently found in these cases. CONCLUSION: The methylation of E-cadherin in oral SCC may occur in the precancerous stage and the process is dynamic, which has no relationship with the aberrant expression of E-cadherin protein.

Paclitaxel affects the amounts of the N-acetylation of 2-aminofluorene and DNA-2-aminofluorene adduct formation in Sprague-Dawley rats.

BACKGROUND: The effects of paclitaxel on the in vivo distribution and the levels of N-acetylation of 2-aminofluorene (AF) and AF-DNA adducts in Sprague-Dawley (SD) rats were studied. METHODS AND RESULTS: For in vivo examination, pretreatment with paclitaxel (50 mg/kg) 48 hours prior to the administration of AF (50 mg/kg) resulted in a 28% and 43% decrease, respectively, in the urinary and fecal recovery of N-acetyl-2-aminofluorene (AAF), and a 22% decrease in the metabolic clearance of AF to AAF. Paclitaxel did not affect the Michaelis-Menten parameters for N-acetyltransferase (NAT) activity in blood, liver, lung, colon and bladder. Similarly, the Km value for AF in the examined tissues was not affected by paclitaxel. However, the Vmax value estimate of liver NAT activity was significantly decreased after paclitaxel pretreatment. Following exposure of rats to AF with and without pretreatment with paclitaxel, DNA-AF adducts were examined in the target tissues, liver, colon and bladder, and also in non-target tissues, lung and circulating leukocytes. The DNA-AF adducts in the liver, bladder, lung, colon and leukocytes were decreased by pretreatment with paclitaxel. CONCLUSION: This is the first finding to show that paclitaxel affects AF distribution and N-acetylation and DNA adduct in SD rats in vivo.

Detection of novel CFTR mutations in Taiwanese cystic fibrosis patients.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) in Asian populations is very rare. We performed molecular genetic analysis in 2 Taiwanese CF patients for detection of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. METHODS: Temporal temperature gradient gel electrophoresis (TTGE) was used for mutation detection, and direct sequencing was used for identification of mutations. RESULTS: In one patient, 2 novel mutations, E7X and 989-992insA, were identified and the carrier status of his parents was confirmed. In the other patient, 3 mutations, S895N, 2215insG, and 1898+5G>T, were found. The 2215insG and S895N were found cis in the same chromosome. These splice site, frameshift, and nonsense mutations produce severely truncated CFTR polypeptides which lack a transmembrane domain, nucleotide binding folds, and the regulatory region, and are predicted to be null in CFTR function. CONCLUSIONS: These cases underscore the importance of comprehensive mutation analysis of Taiwanese CF patients. Definitive molecular findings can confirm the clinical diagnosis and facilitate patient management, carrier testing, and genetic counseling. Furthermore, there is an urgent need to understand the mutation spectrum and the clinical features of the CFTR gene in Asian patients in order that a mutation panel can be established for effective screening of CF chromosomes.

Determination of Di-(2-ethylhexyl)phthalate (DEHP) metabolites in human hair using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Di-(2-ethylhexyl)phthalate (DEHP) is an endocrine disrupting chemical that is widely used as the major plasticizer for worldwide plastic products. It can cause several toxic effects to human with high dose exposure. In response to the need of human exposure assessment, different biological specimens are taken into account. Compared to blood, urine and other specimens, hair is unique in that it could determine the time period of chemical exposure after several months to years. METHOD: The developed method consists of solution incubation, liquid-liquid extraction and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. RESULTS: A reliable and sensitive analytical method was developed and validated for the determination of 5 metabolites, mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxy-hexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono-[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) in human hair. Ten authentic hair specimens were successfully determined and quantitated by the developed method. CONCLUSION: The developed LC-MS/MS method can successfully determine specific DEHP metabolites in human hair and has a great potential to assess the long term DEHP exposure of human.

Honokiol inhibits gastric tumourigenesis by activation of 15-lipoxygenase-1 and consequent inhibition of peroxisome proliferator-activated receptor-gamma and COX-2-dependent signals.

BACKGROUND AND PURPOSE: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), COX-2 and 15-lipoxygenase (LOX)-1 have been shown to be involved in tumour growth. However, the roles of PPAR-gamma, COX-2 or 15-LOX-1 in gastric tumourigenesis remain unclear. Here, we investigate the role of 15-LOX-1 induction by honokiol, a small-molecular weight natural product, in PPAR-gamma and COX-2 signalling during gastric tumourigenesis. EXPERIMENTAL APPROACH: Human gastric cancer cell lines (AGS, MKN45, N87 and SCM-1) were cultured with or without honokiol. Gene and protein expressions were analysed by RT-PCR and Western blotting respectively. Small interfering RNAs (siRNAs) for COX-2, PPAR-gamma and 15-LOX-1 were used to interfere with the expressions of these genes. A xenograft gastric tumour model in mouse was used for in vivo study. KEY RESULTS: PPAR-gamma and COX-2 proteins were highly expressed in gastric cancer cells. Inhibitors, or siRNA for COX-2 or PPAR-gamma, significantly decreased cell viability. Honokiol markedly inhibited PPAR-gamma and COX-2 expressions in gastric cancer cells and tumours of xenograft mice, and induced apoptosis and cell death. Honokiol markedly activated cellular 15-LOX-1 expression and 13-S-hydroxyoctadecadienoic acid (a primary product of 15-LOX-1 metabolism of linoleic acid) production. 15-LOX-1 siRNA could reverse the honokiol-induced down-regulation of PPAR-gamma and COX-2, and cell apoptosis. 15-LOX-1 was markedly induced in tumours of xenograft mice treated with honokiol. CONCLUSIONS AND IMPLICATIONS: These findings suggest that induction of 15-LOX-1-mediated down-regulation of a PPAR-gamma and COX-2 pathway by honokiol may be a promising therapeutic strategy for gastric cancer.

Orthogonal array optimization of microwave-assisted derivatization for determination of trace amphetamine and methamphetamine using negative chemical ionization gas chromatography-mass spectrometry.

An orthogonal array design (OAD) was applied to optimize microwave-assisted derivatization (MAD) for analysis of trace amphetamine (AM) and methamphetamine (MA) by negative chemical ionization gas chromatography-mass spectrometry (NCI GC-MS). The 2,3,4,5,6-pentafluorobenzoyl chloride (PFBC) was used as a derivatization reagent. Experimental factors including solvent, microwave power, and irradiation time at four-levels were studied in 16 trials by OAD(16) (4(4)). The significance of these factors was investigated using analysis of variance (ANOVA) and percent contribution (PC). Solvent is statistically demonstrated a chief factor; microwave power and irradiation time are secondary factors. Under the optimum condition, calibration curve of AM is linear over a range from 0.01 to 100 ng mL(-1) with correlation coefficient 0.9988, and MA from 0.1 to 1000 ng mL(-1) with correlation coefficient 0.9951. The limit of detection (LOD) is 1.20 pg mL(-1) for AM and 13.04 pg mL(-1) for MA. An applicability of the method was tested by analyzing urine samples from amphetamine-type stimulants (ATS)-abusing suspects. Consequently, the OAD method not only optimizes the MAD condition for determination of trace AM and MA, but identifies the effects of factor solvent, microwave power and irradiation time on the MAD performance.

Simultaneous quantitative determination of amphetamines, ketamine, opiates and metabolites in human hair by gas chromatography/mass spectrometry.

A gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the determination of common drugs of abuse in Asia. The method was able to simultaneously quantify amphetamines (amphetamine; AP, methamphetamine; MA, methylenedioxy amphetamine; MDA, methylenedioxymeth mphetamine; MDMA, methylenedioxy ethylamphetamine; MDEA), ketamine (ketamine; K, norketamine; NK), and opiates (morphine; MOR, codeine; COD, 6-acetylmorphine; 6-AM) in human hair. Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol/trifluoroacetic acid (methanol/TFA). The samples were extracted by solid-phase extraction (SPE), derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives were analyzed by electron ionization (EI) GC/MS in selected ion monitoring mode. Confirmation was accomplished by comparing retention times and the relative abundances of selected ions with those of standards. Deuterated analogs of the analytes were used as internal standards for quantification. Calibration curves for ten analytes were established in the concentration range 0.1-10 ng/mg with high correlation coefficients (r2 > 0.999). The intra-day and inter-day precisions were within 12.1% and 15.8%, respectively. The intra-day and inter-day accuracies were between -8.7% and 10.7%, and between -5.9% and 13.8%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) obtained were 0.03 and 0.05 ng/mg for AP, MA, MDA, MDMA and MDEA; 0.05 and 0.08 ng/mg for K, NK, MOR and COD; and 0.08 and 0.1 ng/mg for 6-AM. The recoveries were above 88.6% for all the compounds, except K and NK which were in the range of 71.7-72.7%. Eight hair samples from known polydrug abusers were examined by this method. These results show that the method is suitable for broad-spectrum drug testing in a single hair specimen.