Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

PURPOSE: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). METHODS: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. RESULTS: We found that the recombinant ß-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. CONCLUSIONS: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

Simple and rapid assay method for simultaneous quantification of urinary nicotine and cotinine using micro-extraction by packed sorbent and gas chromatography-mass spectrometry.

A simple and rapid method for determination of nicotine and cotinine levels in urine was developed using samples prepared by micro-extraction by packed sorbent (MEPS) and subjected to gas chromatography-mass spectrometry (GC-MS) analysis. This method provided good reproducibility, as well as good linearity of calibration curves in the range of 1-100 and 50-1000 ng/mL for quality control samples spiked with nicotine and cotinine, respectively. The detection limit of nicotine and cotinine was as low as 0.25 and 20 ng/mL, respectively. An evaporation procedure is not suitable for nicotine determination, thus an advantage of the present MEPS assay method is direct testing with GC-MS without the need for evaporation to a dry solvent. Our findings show that it may be useful for determining nicotine levels in various types of research studies.

Novel extraction method using an ISOLUTE PLD+ protein and phospholipid removal column for liquid chromatography-tandem mass spectrometry analysis of 20 psychoactive drugs in postmortem whole blood samples.

A novel sample extraction method using an ISOLUTE PLD+ protein and phospholipid removal column was developed for simultaneous quantification of 20 psychoactive drugs, including antidepressants, antipsychotics, sedative-hypnotics, and amphetamines, in postmortem whole blood samples by liquid chromatography-tandem mass spectrometry. The method showed improvement in extract cleanliness compared with traditional protein precipitation and the QuEChERS extraction method. The method was validated for all analytes; the calibration curves showed good linearity, with r2 values exceeding 0.991. The intra- and interday accuracies and precisions were 87.6-117.5% and 1.0-18.6%, respectively. The recovery efficiencies were in the range of 64.6-96.8%. Matrix effects were observed in the range of 82.6-116.0%. All analytes were stable under different storage conditions. This method was successfully applied in postmortem forensic sample analysis to quantify psychoactive drugs. The method described in the current study will be useful for forensic toxicological investigations.

High-throughput and simultaneous analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry in the positive and negative ionization modes.

In this report, a high-throughput and sensitive method for analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the positive and negative ionization modes using tolbutamide as internal standard is presented. After pretreatment of a plasma sample by solid-phase extraction with an Oasis HLB cartridge, muscle relaxants were analyzed by UPLC with Acquity UPLC BEH C(18) column and Acquity TQD tandem quadrupole mass spectrometer equipped with an electrospray ionization interface. The calibration curves for muscle relaxants spiked into human plasma equally showed good linearities in the nanogram per milliliter order range. The detection limits (signal-to-noise ratio = 3) was as low as 0.1-2 ng/mL. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with muscle relaxants. To further validate the present method, 250 mg of chlorphenesin carbamate was orally administered to a healthy male volunteer, and the concentrations of chlorphenesin carbamate in plasma were measured 0.5, 1, 2, 4, 6, and 8 h after dosing; their concentrations in human plasma were between 0.62 and 2.44 µg/mL. To our knowledge, this is the first report describing simultaneous analysis of over more than two central-acting muscle relaxants by liquid chromatography-tandem mass spectrometry. This has been realized by the capability of our instrument for simultaneous multiple reaction monitoring of the target compounds in both positive and negative ionization modes. Therefore, the present method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.

Application of Lithium Assay kit LS for quantification of lithium in whole blood and urine.

A commercially available kit for the quantitation of lithium, the Lithium Assay kit LS, was originally developed to measure lithium in serum or plasma using a conventional microplate reader. We investigated whether use of the kit could be extended to quantify lithium in whole blood and urine samples collected at autopsy. The calibration curve for whole blood showed good linearity ranging from 0.5 to 20 µg/mL with a coefficient of determination of 0.998 when samples were pretreated with methanol followed by acetonitrile. Moreover, for urine, we obtained excellent linearity with a coefficient of determination of 0.999 without any pretreatment. The accuracies and precisions were 106.3-174.7% and 1.9-18.1% for whole blood and 83.3-118.8% and 5.7-33.8% for urine. The values in the lower concentration range (0.5-1 µg/mL) were not satisfactory, whereas those in the higher range (2-20 µg/mL) were acceptable. The Lithium Assay kit LS was successfully applied to the measurement of lithium in whole blood and urine samples collected at autopsies. This method appears to be useful for forensic toxicological investigations because of its simplicity and speed.

Frequent detection of noroviruses and sapoviruses in swine and high genetic diversity of porcine sapovirus in Japan during Fiscal Year 2008.

A molecular biological survey on porcine norovirus (NoV) and sapovirus (SaV) was conducted in Toyama Prefecture, Japan, during fiscal year 2008. Both NoV and SaV were detected from swine fecal samples throughout the surveillance period, indicating that these viruses were circulating in this region. NoV strains detected in this study belonged to three genotypes that are known as typical swine NoVs. Although human NoVs were occasionally detected, it was unclear whether they replicated in pigs. As for SaV, genogroup VII (GVII) and other divergent genogroups were identified in addition to the dominant genogroup, GIII, which is the prototypic porcine SaV. In addition, 3 strains genetically related to human SaV were detected. Two of these 3 strains were closely related to human SaV GV. Our study showed that genetic diversification of porcine SaV is currently progressing in the swine population.

Characterization of neutralizing epitopes of varicella-zoster virus glycoprotein H.

Varicella-zoster virus (VZV) glycoprotein H (gH) is the major neutralization target of VZV, and its neutralizing epitope is conformational. Ten neutralizing human monoclonal antibodies to gH were used to map the epitopes by immunohistochemical analysis and were categorized into seven epitope groups. The combinational neutralization efficacy of two epitope groups was not synergistic. Each epitope was partially or completely resistant to concanavalin A blocking of the glycomoiety of gH, and their antibodies inhibited the cell-to-cell spread of infection. The neutralization epitope comprised at least seven independent protein portions of gH that served as the target to inhibit cell-to-cell spread.

Continuous presence of noroviruses and sapoviruses in raw sewage reflects infections among inhabitants of Toyama, Japan (2006 to 2008).

Various genotypes of norovirus (NoV) (genogroup I genotype 1 [GI.1], -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15), and sapovirus (SaV) (GI.1 and GI.2, GII.1, and GIV.1) were detected from raw sewage from April 2006 to March 2008, while limited numbers of genotypes of NoV (GI.8, GII.4, GII.6, and GII.13) and SaV (GII.3 and GIV.1) and of NoV (GII.4, GII.7, and GII.13) were detected from clinical cases and healthy children, respectively. During the winter 2006 to 2008, a large number of sporadic gastroenteritis outbreaks and many outbreaks caused by NoV GII.4 occurred among inhabitants in Toyama, Japan. The copy number of genomes of NoV GII detected from raw sewage changed in relation to the number of outbreaks. NoV strains of the same genotypes observed in both raw sewage and human specimens belonged to the same cluster by phylogenetic analysis and had almost identical nucleotide sequences among each genotype. These data suggest that NoVs and SaVs detected from raw sewage reflect the viruses circulating in the community, irrespective of symptoms, and that subclinical infections of NoV are common in Japan. Combined surveys of raw sewage with those of clinical cases help us to understand the relationship between infection of these viruses and gastroenteritis.

Detection of a novel recombinant norovirus from sewage water in toyama prefecture, Japan.

Recently, the recombination event of norovirus (NoV) has been reported with high frequency, suggesting that RNA recombination is a major driving force in NoV evolution. To assess the incidence of NoV recombination in a residential area, we conducted a molecular biological survey of NoVs existing in sewage water in Toyama Prefecture, Japan. Although GII/4 was predominantly detected in sewage water that was associated with a high frequency of outbreaks caused by this genotype, other genotypes, including two types of recombinant strain, were identified during the survey period. One of the recombinants is the WUG1 type, which was first detected in Saitama Prefecture in 2000. The other recombinant is a novel type derived from two parent strains of genogroup II, GII/7 for the RNA-dependent RNA polymerase and GII/13 for the capsid. This suggests that certain NoVs circulating in the area are occasionally changing their genetic properties by recombination events.

Genetic changes of coxsackievirus A16 and enterovirus 71 isolated from hand, foot, and mouth disease patients in Toyama, Japan between 1981 and 2007.

We characterized the genetic diversity of the complete VP1 region of coxsackievirus A16 (CA16) and enterovirus 71 (EV71) isolated from patients with hand, foot, and mouth disease in Toyama from 1981 to 2007 to evaluate the relationship between epidemics and genetic changes. The predominant genogroups of CA16 changed from B to C in 1995-1998, and genogroup C further changed from subgenogroup C1 to C2 around 2002, and to C3 in 2005-2007. The subgenogroups of the EV71 isolates were classified into B1, B4, C1, and C3 in 1983-1994, and into C4 in 1997-2006. However, changes of the amino acid sequences of the VP1 regions of CA16 were restored, and those of the EV71 isolates were not observed among the same subgenogroups during this survey period, indicating that the prevalence that occurred at intervals of several years seemed to depend on an accumulating number of immunologically naive children, not viral antigenic changes.

Single base substitutions in the capsid region of the norovirus genome during viral shedding in cases of infection in areas where norovirus infection is endemic.

Norovirus (NoV) infections are the major cause of food- and waterborne nonbacterial gastroenteritis in Japan. Some individuals showed long-term excretion of the virus into feces in 29 outbreaks of acute nonbacterial gastroenteritis that occurred in Toyama Prefecture, Japan, in fiscal year 2006. In one of these cases, single base substitutions from A to G in the capsid region of the NoV genome were commonly detected in two individuals during virus shedding by direct sequencing of PCR products. The A-to-G substitution was accompanied by an N-to-S amino acid change. The population of clones that possessed A at the corresponding site was gradually replaced by those with G during the infectious course. Although other substitutions were observed in the complete open reading frame 2 sequence, they were not common in these two individuals. NoVs are capable of evolving in the gastroenteric tract.

A new method for simultaneous quantification of fosphenytoin, phenytoin and its primary metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin in whole blood by ultra-performance liquid chromatography-tandem mass spectrometry.

A method for simultaneous quantification of fosphenytoin (F-PHT), phenytoin (PHT) and its main metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) in whole blood was developed and validated using ultra-performance liquid chromatography-tandem mass spectrometry. Whole blood samples were pretreated by liquid-liquid extraction with acetonitrile and methanol. Chromatographic separation was performed using a CORTECS™ UPLC® C18 (2.1 × 50 mm i.d., particle size 1.6 µm) analytical column, and water containing 10 mM ammonium formate and acetonitrile as the mobile phase. Quantification of the analytes was carried out using mass chromatography with each product ion referenced against phenytoin-d10 as an internal standard. Calibration curves exhibited good linear relationships in a range from 0.005 to 50 µg/ml with correlation coefficients exceeding 0.995. The limits of detection were estimated to be 0.002-0.01 µg/ml. The accuracies and precisions were 96.2-104.3% and 0.7-10.7%, respectively. The recovery efficiencies were in the range of 42.4-59.2%. Matrix effects were observed for PHT and HPPH, with signal suppression ranging from -6.6 to -32.2%. Matrix effect for F-PHT (-5.0 to 8.9%) was less than those for PHT and HPPH. All analytes were stable under different storage conditions. This method was successfully applied for the quantification of F-PHT, PHT and HPPH in rat whole blood samples taken after bolus intravenous administration of F-PHT.

[Epidemiological surveillance for viral pollution of rivers in Toyama Prefecture].

OBJECTIVE: Virus pollution of three rivers in Toyama Prefecture was surveyed over a long period in order to predict and to prevent water-born infection. METHODS: Water samples were collected from three rivers (Itachi, Sembo, Oyabe), then concentrated and inoculated into cultured cells to isolate viruses. The survey at Itachi River was carried out 4 times: in 1979-1981, 1983-1985, 1993-1995, and 2002-2003. The surveys at Sembo and Oyabe Rivers were carried out twice (together with the 3rd and 4th surveys of Itachi River). RESULTS: 1) Various species of enteric viruses, i.e., poliovirus, human enteroviruses B (HEV-B), and reovirus were isolated from Itachi River. Since polioviruses were isolated at the same time as the oral vaccination of babies, these isolates appeared to be derived from vaccine strains. Consistently, isolates in the 3rd and the 4th surveys had only 0-2 base differences in the 480 or 474 nucleotide sequences of the VP3-VP1 region, compared with vaccine strains. The poliovirus detection rates, defined as the ratios of times viruses were detected to the total investigation times, were 33.3%, 41.7%, 2.1% and 0% for the Itachi River from the 1st to 4th surveys, respectively. The lowering between the 2nd and the 3rd surveys was significant (P< 0.001), this being associated with the widespread introduction of paper diapers for babies. The types of HEV-B were various and coincided well with those of prevalent clinical isolates. Reoviruses were frequently detected throughout the year in the 1st and the 2nd surveys, but fell in spring and summer in the 3rd and 4th surveys. 2) The types and the rates of viruses detected from Sembo and Oyabe Rivers were similar to those from Itachi River. CONCLUSIONS: The detection rates of poliovirus and reovirus in the river water during 2002 to 2003 were lower than during 1979 to 1981. This may be due to the improvement of sewerage system or sewerage and the increase in use of paper diapers for babies. The reason that the detection rate of HEV-B did not similarly decrease, and the fact that various types of HEV-B were isolated every year seemed to reflect the epidemic status of HEV-B among inhabitants. Thus, while virus pollution of river water has generally decreased, there is still a possibility of outbreak of water-born infections, since rivers continue to be contaminated with various species of viruses.

Simultaneous quantification of batrachotoxin and epibatidine in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantification of batrachotoxin and epibatidine in plasma. Plasma samples were pretreated by liquid-liquid extraction with acetonitrile and methanol. The toxins were separated on a reversed phase C18-column (2.1mm×50mm, 1.7µm) using a formic acid/acetonitrile gradient elution. Quantification was carried out by mass chromatography with each product ion referenced against midazolam-d4 as an internal standard (IS). The two toxins and the IS were separated within 2min. The calibration curves for the two toxins spiked into human plasma showed good linearities in the range from 2.5 to 250ng/mL. The detection limits were estimated to be 0.5ng/mL for batrachotoxin and 1ng/mL for epibatidine with a signal-to-noise ratio of 3:1. Overall recoveries ranged from 69.6% to 98.2%, and no significant matrix effects were observed. The intra- and interday accuracies were 94.7-102.3%, and the precisions were 1.0-10.3%. This method was successfully applied for the quantification of batrachotoxin and epibatidine in rat plasma samples taken after intraperitoneal administration of the toxins. This is the first report to use UPLC-MS/MS to simultaneously quantify batrachotoxin and epibatidine in plasma samples.

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine.

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography-mass spectrometry.

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.

Development of a poliovirus neutralization test with poliovirus pseudovirus for measurement of neutralizing antibody titer in human serum.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV.

Continuity and change of Japanese encephalitis virus in Toyama Prefecture, Japan.

To determine the mechanisms of maintenance and evolution of Japanese encephalitis virus (JEV) in a temperate zone, we attempted to isolate JEV from mosquitoes and pigs in Toyama Prefecture, Japan. A total of 87 JEVs were isolated from female Culex tritaeniorhynchus mosquitoes and pigs during 2005-2009. The prevalence of JEV in Toyama Prefecture was seasonally late in comparison with that of the virus during 1966-1972. Furthermore, JEVs were isolated after the peak in the number of female Cx. tritaeniorhynchus. Among JEV strains isolated in this study, two distinct groups were observed within genotype I of the phylogeny generated from nucleotide sequence information derived from the envelope and capsid/premembrane genes: strains belonging to the major type were isolated during 2005-2009, and strains from the minor type were isolated only in 2007. The major type has exhibited gradual change in its envelope and capsid/premembrane genes, and all isolates obtained in 2008 and 2009 had a novel deletion of seven nucleotides in the variable region of the 3'-untranslated region.

Widespread circulation of echovirus type 13 demonstrated by increased seroprevalence in Toyama, Japan, between 2000 and 2003.

To confirm the magnitude of an echovirus type 13 (E13) outbreak in 2002 and to evaluate whether genetic and antigenic changes in E13 influenced the occurrence of the outbreak, we measured titers of neutralizing (NT) antibody against the Toyama, 2002-240-SF, and prototype Del Carmen E13 strains among inhabitants of Toyama before and after 2002. The rate of positivity for NT antibodies against both 2002-240-SF and Del Carmen in 2003 made a remarkable upturn in children 0 to 14 years old, compared to that in 2000. Titers of NT antibody against strain 2002-240-SF of inhabitants were slightly higher than those against Del Carmen, whereas anti-E13 rabbit serum raised against either strain Del Carmen or 2002-240-SF showed almost the same titer of NT antibody against both strains. These data indicate that the antigenic properties of the strains may be slightly different. Differences in amino acids between strains 2002-240-SF and Del Carmen in the VP4, VP2, VP3, and VP1 regions may affect both antigenic and receptor binding properties, even though they do not seem to be significant enough to escape widespread immunity. One of the factors of the outbreak was thought to be the increase in susceptibility in the young generation.