Development of a method to evaluate the effects of external environments on drug stability in nails using micro-segmental analysis.

Nails can be used as an alternative to hair for examining past drug use. However, daily hand-and-nail care can eliminate the internal drugs. Therefore, we developed an evaluation method to examine the effects of the external environment on drug stability in nails using micro-segmental analysis. First, reference nails containing drugs were prepared by collecting fingernails from participants who had consumed hay-fever medicines continuously for 4 months. Next, the entire free edge of a reference nail was cut into halves at the centerline; one side was stored as an untreated block, and the other was treated with various hand/nail care products. Both nail blocks were washed and segmented at 0.5-mm intervals in the width direction. Each segment in the extraction solution was crushed with stainless-steel beads, sonicated, and soaked in the solution for 24 h. The analytes in extracts were quantified by LC-MS/MS, and the drug concentrations between the treated and untreated blocks were compared. The drug concentrations decreased slightly in nails treated with manicure and gel-nail products. The analytes in nails tended to be lower in water-rich products such as hand soap and hand cream than in oil-rich products such as nailcare oil and acetone-free remover. The developed method using micro-segmental analysis enabled the evaluation of the effects of various hand/nail care products on drug stability in a limited number of nails. This would also be useful for examining the effects of severe environments on drugs in nails collected from cases of unnatural death.

Metabolism of highly potent synthetic opioid nitazene analogs: N-ethyl-N-(1-glucuronyloxyethyl) metabolite formation and degradation to N-desethyl metabolites during enzymatic hydrolysis.

The metabolism of the highly potent synthetic opioids metonitazene, etonitazene, and protonitazene was investigated in fresh human hepatocytes. In the hydrolyzed culture medium, N-desethyl-, N,N-di-desethyl-, O-desalkyl-, N-desethyl-O-desalkyl-, N,N-di-desethyl-O-desalkyl-, and N-oxidated metabolites were detected as phase I metabolites, whereas in the unhydrolyzed culture medium, O-glucuronides of phase I metabolites with O-dealkylation were detected as phase II metabolites. The detected phase I metabolites were identified by comparing their analytical data with those of synthesized authentic standards. In contrast, phase II metabolites were identified by comparing their analytical data with those of the glucuronidated products formed by the incubation of the corresponding substrates with human liver microsomes in the presence of uridine diphosphate glucuronic acid. In addition to the aforementioned metabolites, some putative N-ethyl-N-(1-glucuronyloxyethyl) metabolites were detected in the unhydrolyzed culture medium. Purification and hydrolysis experiments revealed that N-ethyl-N-(1-glucuronyloxyethyl) metabolites formed the corresponding N-desethyl metabolites via unstable N-ethyl-N-(1-hydroxyethyl) metabolites during enzymatic hydrolysis.

Identifying a suspect powder as a cannabis concentrate through chemical analysis and DNA testing.

PURPOSE: Cannabis is regulated in many countries, and cannabis products are diversifying, which can hinder identification. Here, we report the seizure of a powder sample with a cannabis-like odor in a spice bottle labeled "nutmeg" and identification of the sample by chemical testing and cannabis DNA testing. METHODS: The sample was observed under a microscope, extracted with methanol, and analyzed by gas chromatography-mass spectrometry (GC-MS). The chemical profile of the seized powder was compared with that of nutmeg samples. Gas chromatography-flame ionization detection was used to estimate the total Δ9-tetrahydrocannabinol (Δ9-THC) concentration in the sample. A commercially available cannabis DNA testing kit was used to confirm the presence of cannabis plant DNA in the seized sample. RESULTS: The characteristics of cannabis in the seized powder were difficult to determine through microscopic observation alone. GC-MS analysis identified ß-caryophyllene (an aromatic component of cannabis) and five cannabinoids unique to cannabis, including Δ9-THC. No common compounds were identified in the seized powder or nutmeg samples. The total Δ9-THC concentration in the sample was very high (approximately 47% by weight). Cannabis DNA testing confirmed that the seized powder contained cannabis. CONCLUSIONS: The seized powder was found to be a processed product made from a finely pulverized resin-like cannabis concentrate. Our results indicate that combined chemical and DNA analysis should help identify cannabis-related samples in various forms.

Effects of temperature, humidity, light, and soil on drug stability in hair: a preliminary study for estimating personal profiles using micro-segmental analysis of corpse hair.

PURPOSE: Micro-segmental hair analysis (MSA), which enables detailed measurement of the distribution of drugs in a single hair strand, is useful for examining the day of death and drug use history of a person. However, corpses are often found in severe environments, such as soil and freezers, which affect the drug contents in hair. Therefore, we examined the effects of temperature, humidity, light, and soil on drug stability in hair as a preliminary study to estimate personal profiles using MSA of corpse hair. METHODS: Four hay-fever medicines (fexofenadine, epinastine, cetirizine, and desloratadine) were used as model drugs to evaluate drug stability in hair. Reference hair strands consistently containing the four medicines along the hair shaft were collected from patients with hay-fever who ingested the medicines daily for 4 months. The hair strands were placed in chambers with controlled temperatures (- 30 to 60 °C) and relative humidities (ca. 18 % and > 90 %), exposed to light (sunlight and artificial lights) or buried in soil (natural soil and compost). RESULTS: Sunlight and soil greatly decomposed the hair surfaces and decreased the drug contents in hair (up to 37 %). However, all analytes were successfully detected along the hair shaft, reflecting the intake history, even when the hair was exposed to sunlight for 2 weeks and buried in the soil for 2 months. CONCLUSIONS: Although the exposure to sunlight and storage in soil for long times made drug-distribution analysis difficult, MSA could be applied even to hair strands collected from corpses left in severe environments.

Identification of 1-(thiophene-2-carbonyl)-LSD from blotter paper falsely labeled "1D-LSD".

PURPOSE: Since the mid-2010s, lysergic acid diethylamide (LSD) analogs made for substance abuse have periodically emerged. In this case, three pieces of blotter paper labeled "1D-LSD" and presumably impregnated with this LSD analog, were seized. Several websites indicate that 1D-LSD is 1-(1,2-dimethylcyclobutane-1-carbonyl)-LSD. Because this analog is much more difficult to synthesize than previously reported LSD analogs, we doubted that the blotter paper contained 1D-LSD. Herein, we determined the structure of the absorbed compound. METHODS: One of the seized specimens was extracted and analyzed using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance (NMR) spectroscopy to estimate the extract components. The estimated compound was then synthesized, yielding an authentic standard. The contents of the seized specimens were identified using authentic standard analysis with GC/MS, LC/MS, and NMR spectroscopy. RESULTS: Instrumental analyses confirmed the active compound to be 1-(thiophene-2-carbonyl)-LSD, which was inconsistent with the labeling on drug-infused blotter paper. CONCLUSION: As in this case, similar blotter paper analyses should consider the possibility of a mismatch between the label and ingredient. To the authors' knowledge, this is the first case report in which 1-(thiophene-2-carbonyl)-LSD was seized and the first seizure of an LSD analog in which an aromatic carboxylic acid had been condensed to LSD. This type of lysergamide may become prevalent in the near future, and we should remain alert for newly appearing lysergamides.

Preparation of glucuronides using liver microsomes and their characterization by 1D/2D NMR spectroscopy and mass spectrometry: Application to fentanyl metabolites.

A simple, low-cost method for preparing glucuronic acid-conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid-conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4'-hydroxy-fentanyl and ß-hydroxy-fentanyl) with uridine 5'-diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4'-hydroxy-fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of ß-hydroxy-fentanyl. Submilligram amounts of three of these metabolites, namely, 4'-hydroxy-fentanyl glucuronide and two glucuronides of ß-hydroxy-fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed-phase/anion-exchange mixed-mode solid-phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6-69%.

Changes in methamphetamine impurity profiles induced by tert-butoxycarbonylation.

Analysis of impurities in methamphetamine (MA) can be used to characterize MA seizures, investigate the relationship among MA seizures, and provide information on their synthetic routes. Recently, chemically derivatized MA, such as tert-butoxycarbonyl (t-Boc) MA, has been seized and attracted attention because routine forensic analysis methods may fail to correctly identify them. Chemical derivatization is a simple method for protection and deprotection of a compound, and protection of MA using t-Boc can be used to mask the MA. Although t-Boc derivatization might alter the impurity profile of MA, the actual changes in the impurity profile have not been investigated. In this study, changes in the MA impurity profile with tert-butoxycarbonylation were explored. MA and some typical impurities were derivatized using di-tert-butyl dicarbonate and water. Analysis of the impurities in five MA samples by gas chromatography showed that peaks both appeared and disappeared for the deprotected MA compared with the original MA. However, typical impurities important for characterizing MA seizures were conserved after derivatization and deprotection. Most of the new peaks were speculated to be contaminants introduced during derivatization and deprotection. A peak giving a mass spectrum similar to that of t-Boc MA was detected in the chromatograms of t-Boc MA and deprotected MA. Although the origin of this peak was not determined, it might be a marker for the MA involving tert-butoxycarbonylation. These results indicate that tert-butoxycarbonylation can alter the MA impurity profile; therefore, care is needed when interpreting results for derivatized MA.

Rapid enantiomeric analysis of zopiclone in serum by supercritical fluid chromatography-tandem mass spectrometry.

Zopiclone (ZOP) is a hypnotic drug prescribed to treat insomnia. Due to the chiral nature of ZOP, the psychologically active S-form and inactive R-form need to be determined enantiomerically in a forensic drug analysis. In the present study, a supercritical fluid chromatography (SFC) method was designed with a faster analysis ability than that of previously reported techniques. The SFC-tandem mass spectrometry (SFC-MS/MS) method was optimized using a column with a chiral polysaccharide stationary phase (Trefoil CEL2). ZOP was extracted from pooled human serum using solid-phase extraction (Oasis HLB) and analyzed. The developed SFC-MS/MS method achieved the baseline separation of S-ZOP and R-ZOP within 2 min. The fit-for-purpose method validation indicated that the optimized solid-phase extraction achieved near complete recovery and approximately 70% of the matrix effect. Both the retention time and peak area showed sufficient precision. The lower and upper limits of quantification (LOQ) were 5.7 × 10-2 ng/mL and 25 ng/mL for R-ZOP, and 5.2 × 10-2 ng/mL and 25 ng/mL for S-ZOP. The calibration line was linear in the range from lower LOQ to upper LOQ. The stability test indicated that ZOP in serum stored in a refrigerator (4°C) degraded and about 55% remained in 31 days. The quick analysis of the SFC-MS/MS method makes it a valid option for the enantiomeric analysis of ZOP.

Evaluation of applicability of micro-segmental analysis to hair treated with heat and haircare products.

PURPOSE: Micro-segmental analysis (MSA), which enables the measurement of detailed drug distributions in hair by segmenting a single hair strand at 0.4 mm intervals, is indispensable for estimating the day of drug ingestion. However, haircare with dryers and various products can influence drug concentrations in hair. Therefore, the applicability of MSA to hair that was treated with heat or various haircare products was evaluated. METHODS: Reference hair strands containing drugs consistently along the hair shafts were collected from patients who ingested four hay-fever medicines (fexofenadine, epinastine, cetirizine, and loratadine) daily for 4 months. The hair strands were divided into eight 4 mm regions from the proximal end, and each region was placed on an electric hot plate at 100-200 °C or soaked in haircare products, such as shampoo and bleaching agent. The hair regions were subjected to MSA. Moreover, after a patient was administered midazolam at a single dose and the hair was bleached, the day of midazolam administration was estimated using MSA. RESULTS: Repetitive heating for 1 min and daily haircare products, such as shampoo, hardly affected the drugs in hair, whereas bleaching products containing H2O2 decreased the amounts of hay-fever medicines in the hair up to 58%. However, the amount of midazolam did not decrease in bleached hair and the day of midazolam administration was successfully estimated. CONCLUSIONS: The analytes used in this study were minimally affected by ordinary haircare and could be detected even in bleached hair. Therefore, MSA can be applicable regardless of haircare history.

Evaluation of decarboxylation efficiency of Δ9-tetrahydrocannabinolic acid and cannabidiolic acid by UNODC method.

PURPOSE: Decarboxylation of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) to Δ9-tetrahydrocannabinol (Δ9-THC) by heating is a common method for determining total Δ9-THC. In the manual for cannabis identification and analysis, the United Nations Office on Drugs and Crime (UNODC) proposed decarboxylation conditions. Although the manual's primary analytical target is Δ9-THC, some reports also quantified cannabidiol (CBD). The authors assessed the efficiency of decarboxylation of Δ9-THCA and cannabidiolic acid (CBDA), a carboxylated form of CBD, under four decarboxylation conditions, including the UNODC condition. METHODS: Δ9-THCA and CBDA were heated in 2-mL glass vials at 150 °C for 12 min after the following treatment: condition A involves the addition of ethanol without capping, condition B involves non addition of solvent without capping, condition C involves non addition of solvent with capping, and condition D (UNODC condition) involves the addition of 0.5 mg/mL tribenzylamine (TBA) in ethanol without capping. The residue after heating was dissolved in methanol and then analyzed by high-performance liquid chromatography. RESULTS: The production of Δ9-THC and CBD was low (≤ 10.1%) under conditions A and B. Under condition C, Δ9-THC production was increased (53.4%), but CBD production was hardly improved (11.7%). Under condition D, Δ9-THC and CBD production dramatically increased to 83.2 and 71.0%, respectively. CONCLUSIONS: These findings indicated that TBA improved the production of Δ9-THC and CBD from their carboxylated forms; however, even in the presence of TBA, their production did not reach 100%. Forensic toxicologists should understand the effectiveness and limitations of decarboxylation under the UNODC condition.

Analysis of highly potent synthetic opioid nitazene analogs and their positional isomers.

Four nitazenes (metonitazene, etonitazene, protonitazene, and isotonitazene), highly potent benzimidazole synthetic opioids, and their four nitro group positional isomers (isonitazenes) were synthesized and analyzed using infrared (IR) spectroscopy, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS). In addition, the agonistic activity of all compounds at the human µ-opioid receptor was measured using a cell-based assay system. In the IR spectra, characteristic peaks for nitazenes and isonitazenes were observed. In GC/MS, all compounds were well separated on the chromatogram, although distinguishing nitazenes from the corresponding isonitazenes by electron ionization mass spectra was difficult. In LC/MS, all compounds were detected in both positive and negative modes of electrospray ionization. Characteristic fragment ions were observed in the product ion spectra of isonitazenes, enabling nitazenes to be distinguished from isonitazenes. All nitazenes tested demonstrated higher agonistic activity at the human µ-opioid receptors than the synthetic opioid fentanyl. The agonistic activities of isonitazenes were 11-35 times lower than those of the corresponding nitazenes. However, iso-etonitazene and iso-isotonitazene showed moderate activity similar to that of fentanyl, indicating that these drugs could cause poisoning at a comparable level as fentanyl, if these drugs are abused in the future.

Possibility of drug-distribution measurement in the hair of drowned bodies: evaluation of drug stability in water-soaked hair using micro-segmental analysis.

In postmortem examinations, the drug analysis of hair is effective for revealing drug-use history. Additionally, a method to estimate the day of death using hair was previously developed by analyzing a single hair strand segmented at 0.4-mm intervals (micro-segmental hair analysis). However, for drowned bodies, drugs in the hair may be washed out due to soaking in water for extended periods. To evaluate the possibility of measuring drug distribution in the hair of drowned bodies, drug stability in hair samples soaked in various aqueous solutions was examined. First, reference hair strands of drug users containing specific drugs consistently along the hair shaft were prepared. The participants ingested 4 hay-fever medicines (fexofenadine, epinastine, cetirizine, and loratadine) every day for approximately 4 months before hair collection. Each reference strand was divided into regions, and each region was soaked in different solutions containing various solutes for extended periods up to approximately 2 months. In solutions without divalent ions (Ca2+ and Mg2+), the drug content in the hair decreased up to approximately 5 % with increasing salt concentration and soaking time. However, the decreased drug content was negligible in solutions containing divalent ions, implying that the divalent ions prevented drugs contained in hair from washing out. As natural river and sea waters contain divalent ions, the drugs in hair were hardly washed out even when the hair was soaked for 2 months. Thus, it was concluded that drug-distribution measurements using micro-segmental analysis can also be applied to the hairs of drowned bodies.

Comments on "discrepancies between validated GC-FID and UHPLC-DAD methods for the analysis of Δ-9-THC and CBD in dried hemp flowers".

The original article described difference of total Δ9 -THC contents determined by gas chromatography with flame ionization detection (GC-FID) and high-performance liquid chromatography with ultraviolet detection (HPLC-UV). We presumed that this difference was mainly caused by peak overlapping of Δ9 -THC and presumptive cannabielsoin (CBE) isomer on the GC chromatogram.

Micro-segmental hair analysis: detailed procedures and applications in forensic toxicology.

PURPOSE: Since the 1980s, the detection sensitivity of mass spectrometers has increased by improving the analysis of drugs in hair. Accordingly, the number of hair strands required for the analysis has decreased. The length of the hair segment used in the analysis has also shortened. In 2016, micro-segmental hair analysis (MSA), which cuts a single hair strand at a 0.4-mm interval corresponding to a hair growth length of approximately one day, was developed. The advantage of MSA is that the analytical results provide powerful evidence of drug use in the investigation of drug-related crimes and detailed information about the mechanism of drug uptake into hair. This review article focuses on the MSA technique and its applications in forensic toxicology. METHODS: Multiple databases, such as SciFinder, PubMed, and Google, were utilized to collect relevant reports referring to MSA and drug analysis in hair. The experiences of our research group on the MSA were also included in this review. RESULTS: The analytical results provide a detailed drug distribution profile in a hair strand, which is useful for examining the mechanism of drug uptake into hair in detail. Additionally, the analytical method has been used for various scenarios in forensic toxicology, such as the estimation of days of drug consumption and death. CONCLUSIONS: The detailed procedures are summarized so that beginners can use the analytical method in their laboratories. Moreover, some application examples are presented, and the limitations of the current analytical method and future perspectives are described.

Thin-layer chromatography on silver nitrate-impregnated silica gel for analysis of homemade tetrahydrocannabinol mixtures.

PURPOSE: Various forms of cannabidiol (CBD)-containing products are sold in Japan. CBD is easily converted to mixtures of ∆9-tetrahydrocannabinol (∆9-THC) and its isomer, ∆8-THC, using household chemicals like diluted hydrochloric acid. This ease of production increases concerns regarding production of homemade THC mixtures. It is difficult to separate ∆9-THC, ∆8-THC, and CBD using thin-layer chromatography (TLC) on conventional silica gel. The selectivity of TLC on silver nitrate-impregnated silica gel (AgNO3-silica gel) differs from that of conventional silica gel. This study thus aimed to evaluate the separation ability of AgNO3-silica gel TLC. METHODS: To evaluate potential separation ability, standards of five THC isomers (∆9-THC, ∆8-THC, a pair of diastereomers of ∆10-THC, and ∆6a,10a-THC), CBD, CBN, and ∆9-THCA were analyzed by 10% AgNO3-silica gel TLC (developed using toluene, system A) and silica gel TLC [developed using n-hexane/diethyl ether (8:2, v/v), system B]. Then, mock homemade THC mixtures, prepared by heating crystalline CBD in acidic ethanol, were analyzed using systems A and B. RESULTS: System A showed clear separation between the five THC isomers and between ∆9-THC, ∆8-THC, CBD, and their by-products in the mock homemade THC mixture. However, system B did not separate some combinations of THC isomers and gave a single group-like spot to the THC mixture. CONCLUSION: AgNO3-silica gel TLC shows high separation ability between THC isomers and among ∆9-THC, ∆8-THC, and CBD. It will thus be useful for analyzing homemade THC mixtures.

Distribution profiles of diphenhydramine and lidocaine in scalp, axillary, and pubic hairs measured by micro-segmental hair analysis: good indicator for discrimination between administration and external contamination of the drugs.

PURPOSE: Drug distribution in scalp hair can provide historical information about drug use, such as the date and frequency of drug ingestion. We previously developed micro-segmental hair analysis, which visualizes drug distribution at 0.4-mm intervals in individual hairs. The present study examines whether the distribution profiles of drugs can be markers for the administration or external contamination of the drugs using scalp, axillary, and pubic hairs. METHODS: A single dose of anti-itch ointment containing diphenhydramine (DP) and lidocaine (LD) was topically applied to the axillary or pubic areas of two volunteers; DP was also orally administered; and LD was intra-gingivally injected. Scalp, axillary, and pubic hairs were assessed using our micro-segmental analysis. RESULTS: The localization of DP and LD differed within individual scalp hair strands, implying DP and LD were predominantly incorporated into scalp hair via the bloodstream and via sweat/sebum, respectively, showing double-peak profiles. However, DP and LD were distributed along the shafts of axillary and pubic hairs without appearance of the double-peak profiles when the ointment had been applied to the axillary and pubic areas. The distributions of DP and LD in scalp hairs did not significantly differ according to administration routes, such as oral administration, gingival injection, and topical application. CONCLUSIONS: Micro-segmental analysis revealed differences in the distribution profiles of drugs in hairs, and distinguished hairs with and without external contamination. These findings will be useful for understanding of the mechanism of drug uptake into hair and for estimating the circumstances for a drug use.

Agonistic activity of fentanyl analogs and their metabolites on opioid receptors.

PURPOSE: This study aims to expose the toxicity of fentanyl analogs and their metabolites by measuring the agonistic activity of these compounds on opioid receptors. METHODS: The agonistic activity of fentanyl, four analogs of fentanyl (acetylfentanyl, butyrylfentanyl, tetrahydrofuranylfentanyl, and furanylfentanyl), and their metabolites were evaluated using a cell-based assay system, which measured the cellular cAMP level after the reaction of a test compound with cells expressing opioid receptor. RESULTS: Fentanyl and its four analogs showed agonistic activity on µ-opioid receptor at < 10 nM, whereas these compounds were inactive at δ- and κ-opioid receptors even at 100 nM. Similarly, no metabolites showed agonistic activity on δ- and κ-opioid receptors. Meanwhile, several metabolites were active at µ-opioid receptor. ß-Hydroxy metabolites exhibited strong activity nearly equivalent to those of the parent drugs. Some 4'-hydroxy metabolites and N-acyl group-hydroxylated metabolites were still active; however, their activity drastically decreased compared to the parent drugs. CONCLUSIONS: Most of the metabolic reactions drastically diminish the agonistic activity of fentanyl analogs; exceptionally, ß-hydroxylation maintains the activity at a level nearly equal to that of the parent drugs. However, ß-hydroxy metabolites should contribute less to the poisoning caused by the ingestion of fentanyl analogs.

Thermal decomposition of CBD to Δ9-THC during GC-MS analysis: A potential cause of Δ9-THC misidentification.

On the analysis of cannabidiol (CBD) e-liquid by gas chromatography-mass spectrometry, we experienced suspected thermal decomposition of CBD to Δ9-tetrahydrocannabinol (Δ9-THC). To clarify the factors involved in the decomposition, we evaluated the effects of the injection methods (splitless or split), injector temperatures (250, 225, 200, and 180 °C), and liner conditions (new liner or used liner) on the CBD decomposition. We also examined whether addition of methylamine to the dissolving solvent (methanol) inhibited the decomposition. Decomposition was not observed under split mode. However, under splitless mode, we observed that decomposition was promoted with the use of used liner and by high injector temperatures, and addition of methylamine to the dissolving solvent also suppressed the decomposition. Split injection was effective for preventing the decomposition; however, splitless injection enables detection of lower-concentrated Δ9-THC in CBD products than split injection. To balance sensitivity of Δ9-THC and inhibition of the thermal decomposition under splitless mode, we recommend using new liner for the analysis, addition of methylamine to the dissolving solvent, and maintenance of the injector temperature at 200 °C.