Tandem Mass Spectrometry for the Analysis of Plasma/Serum Acylcarnitines for the Diagnosis of Certain Organic Acidurias and Fatty Acid Oxidation Disorders.

Acylcarnitines are formed in the mitochondria by esterification between carnitine and acyl-CoAs. This occurs enzymatically via carnitine acyltransferases. Specific acylcarnitines accumulate as a result of various organic acidurias and fatty acid oxidation disorders, and, thus, acylcarnitines profiles are used for the diagnosis of these disorders. Acylcarnitines monitoring can also be used for the follow-up of patients with these disorders. Tandem mass spectrometry (MS/MS) is the most commonly used method for the analysis of acylcarnitines. An MS/MS method for the quantification of a number of acylcarnitines is described. The method involves butylation of acylcarnitines using acidified butanol. Butylated acylcarnitines are analyzed using flow injection and precursor ion scan. Multiple-reaction monitoring (MRM) is used for the analysis of low-molecular-weight acylcarnitines.

Quantification of Free and Total Carnitine in Serum Using Liquid Chromatography Tandem Mass Spectrometry.

L-carnitine is a crucial component for transporting long-chained fatty acids from the cytosol into the mitochondrial matrix for fatty acid oxidation. During this process, carnitine forms numerous acylcarnitines before being recycled into the cytosol. Abnormal levels of free carnitine, total carnitine, and acylcarnitines in serum can be indicative of a metabolic disorder before symptoms are present. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is described for the determination of free and total carnitine in serum. To measure total carnitine, samples are spiked with deuterated carnitine (internal standard) and hydrolyzed with potassium hydroxide to convert acylcarnitines to carnitine. The reaction is quenched by the addition of hydrochloric acid. Carnitine is extracted via a methanolic protein precipitation. The solution is then injected on LC-MS/MS for analysis to determine the carnitine concentration using multiple-reaction monitoring.

Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chromatography-Mass Spectrometry (GC/MS).

Neuroblastoma and other neural crest tumors can be characterized by the increased production and excretion of catecholamines and their metabolites. Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are important catecholamine metabolites that can be measured to provide relatively rapid laboratory diagnosis and clinical follow-up of neuroblastoma. We present a procedure to quantify HVA and VMA in urine samples which have been diluted to a creatinine concentration of 2 mg/dL. Diluted samples are spiked with deuterated internal standards, acidified, and extracted with an organic solvent. A bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine mixture is added to the dried extract to create trimethylsilyl derivatives of HVA and VMA. The derivatized compounds are measured using gas chromatography-mass spectrometry (GC/MS).

Screening of Organic Acidurias by Gas Chromatography-Mass Spectrometry (GC-MS).

Organic acidurias or acidemias are a group of diverse disorders caused by decreased or diminished activity of specific enzyme or transporter involved in the metabolism of amino acids, carbohydrates, fatty acids, and nucleic acids. Organic acidurias are generally inherited but may be acquired due to deficiency of certain cofactors or vitamins. As clinical symptoms are of nonspecific nature, definitive diagnosis of organic aciduria requires measurement of organic acids in urine or blood and sometimes enzyme activity in the cells. Gas chromatography-mass spectrometry (GC-MS) is a commonly used method for screening of organic acidurias.GC-MS procedure described here involves the use of urine volume that contains 1 µmole (113 µg) of creatinine. Internal standards (tropic and 2-ketocaproic acids) are added to the samples, followed by treatment with hydroxylamine to form oxime derivatives of the ketoacids. The mixture is then acidified, and organic acids are extracted in ethyl acetate. The organic extract is concentrated to dryness, and the residue is treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS)/pyridine to form the trimethylsilyl (TMS) derivatives of the organic acids. The derivatized extract is then directly injected onto GC-MS for analysis.

Quantitation of Phenylalanine in Dried Blood Spot Using Liquid Chromatography Tandem Mass Spectrometry for Monitoring of Patients with Phenylketonuria (PKU).

Newborn screening for phenylketonuria (PKU) is performed by analysis of phenylalanine in dried blood spot (DBS). Once diagnosed by a definitive method, a patient's dietary control is performed by repeated analysis of phenylalanine in venous blood or DBS. Since venipuncture is time consuming, painful, and may often be difficult to achieve in newborns, the use of DBS for analysis of phenylalanine is becoming a preferred method for dietary monitoring of patients with PKU. Using a lancet, patients or their guardians collect finger capillary blood on an approved filter paper. Once collected, the filter paper with DBS is sent to the laboratory for phenylalanine analysis. In the laboratory, phenylalanine is extracted from the DBS using organic solvents. Here, we describe an LC-MS/MS method for the analysis of phenylalanine from DBS with an approximation to serum levels.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Steroids Androstenedione, Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone.

Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive disorders due to deficiency of 11- or 21-hydroxylase. The analysis of cortisol, androstenedione, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Analysis of specific steroids is also performed in other disorders such as evaluation of hirsutism or infertility in females and hypogonadism in males. Cortisol is generally analyzed by immunoassays, whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC-MS/MS method is described for the simultaneous quantification of androstenedione, 17-hydroxyprogesterone, DHEA, 11-deoxycortisol and testosterone. The method involves addition of labeled internal standards to serum samples and extraction of steroids in methyl tert-butyl ether. The extract is evaporated under stream of nitrogen, and the residue is reconstituted in methanol and analyzed by LC-MS/MS.

Quantification of Tryptophan, Indole, and Indoxyl Sulfate in Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Having a diverse gut microbiota has been correlated with the short- and long-term success of allogeneic stem cell transplantation. Intestinal bacteria metabolize the amino acid tryptophan to indole. Indole is further oxidized and sulfonated in the liver to 3-indoxyl sulfate (3-IS), which is then excreted in urine. Urinary 3-IS is a potential biomarker for intestinal health and an early predictor of successful stem cell transplantation. We describe a rapid method for quantifying tryptophan, indole, and 3-indoxyl sulfate in urine specimens, in which urine samples are diluted with a formic acid solution and deuterated internal standards, and then injected on LC-MS/MS for analysis.

Quantification of 25-Hydroxyvitamin D2 and D3 Using Liquid Chromatography-Tandem Mass Spectrometry.

Vitamin D plays an important role not only in bone health but also in many other body functions. Vitamin D deficiency is very common in the general population. Measurement of blood 25-hydroxyvitamin D is a common practice to evaluate vitamin D deficiency. Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are the most commonly used methods for the measurement of 25-hydroxyvitamin D. Immunoassays suffer from specificity issues and do not distinguish between 25-hydroxyvitamin D2 and D3. Therefore, LC-MS/MS is a preferred method for quantification of 25-hydroxyvitamin. We describe an LC-MS/MS method, which involves protein precipitation and analysis of the extract using atmospheric pressure chemical ionization and multiple reaction monitoring. 25-hydroxyvitamin D3-d6 is used as an internal standard. The method is linear from 1-100 ng/mL for both 25-hydroxyvitamin D2 and D3 and has imprecision of <10%.

Analysis of U-47700, a Novel Synthetic Opioid, in Human Urine by LC-MS-MS and LC-QToF.

The illicit drug market has rapidly evolved from synthetic cannabinoids to cathinone derivatives and now a new emerging threat of synthetic opioids. These compounds were mostly developed by pharmaceutical companies during drug discovery. The new psychoactive substances are not routinely covered in drug screening and may go undetected. Recently fentanyl analogous, AH-7921, MT-45 and now U-47700 have been encountered in clinical and forensic casework. U-47700 is gaining popularity on drug user forms as a legal alternative to heroin. It is a µ-receptor agonist that is part of the trans-1-2-diamine opioid analgesic drug class developed by The Upjohn Company in an attempt to develop a non-addicting analgesic. A LC-MS-MS method was developed and validated to detect and quantify U-47700. Additional analysis was conducted with an LC-QToF to identify the presence of the parent drug and metabolites. A total of four cases have been evaluated by the LC-MS-MS methodology which has an analytical range of 1-1,250 ng/mL and limit of detection of 1 ng/mL. The concentration of U-47700 in urine specimens ranged from below the limit of quantification to 224 ng/mL. The ToF analysis detected the presence of suspected phase I demethylated metabolites that may assist future analysis of this compound. The prevalence of designer opioids in casework highlights the importance of analysis for new psychoactive substances. Traditional opiates/opioids were not detected in the presented cases, but the available case histories revealed an opioid toxidrome. These findings suggest that U-47700 drug may cause significant morbidity and mortality within the United States as an emerging drug threat.

Two cases of intoxication with new synthetic opioid, U-47700.

BACKGROUND: Novel substances often referred to as "designer drugs" have emerged as drugs of abuse, and recognition of these is difficult as routine blood and urine screening tests do not detect these agents. U-47700 is a synthetic selective µ-opioid agonist that can be bought online for as little as $40 per gram. We report two patients presenting after insufflation of U-47700, with subsequent confirmation of this substance in urine samples. CASE DETAILS: A 26-year-old man and 24-year-old woman insufflated a substance they believed to be "synthetic cocaine." The man was found down with cyanosis and agonal respirations. He was intubated and taken to hospital where he recovered well with supportive care. The woman presented with anxiety, tremors and drowsiness and was admitted for observation. Urine samples from both patients were analyzed using GC/MS/MS and LC/QToF, and U-47700 was isolated in both cases. No other opioids were detected. DISCUSSION: These cases are concerning because U-47700 is a relatively new agent that is easy to obtain over the internet and has the potential to cause significant morbidity and mortality.

Simultaneous determination of cyclosporine, sirolimus, and tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry.

A multiple reaction monitoring, positive ionization electrospray, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous quantification of cyclosporine, sirolimus, and tacrolimus in human whole blood. Proteins in the samples are precipitated with a mixture of methanol and zinc sulfate. The supernatant is injected into the LC-MS/MS for analysis. Chromatography involves the use of a C18 column and ammonium acetate/water/methanol-containing mobile phases. The MS/MS is operated in positive ion electrospray mode. Quantification is achieved by comparing peak area ratios of MRMs of analytes and internal standards with that of calibrators. Calibration curves are constructed from peak area ratios of MRMs of calibrators and internal standards versus concentrations. MRMs used are ascomycin (m/z 809.5 → 756.5), cyclosporine A (m/z 1,219.9 → 1,203.1), cyclosporine D (m/z 1,234.0 → 1,217.0), sirolimus (m/z 931.6 → 864.5), and tacrolimus (m/z 821.5 → 768.4).

Three fatal intoxications due to methylone.

We present three fatal intoxications of methylone, a cathinone derivative. Blood was analyzed with a routine alkaline liquid-liquid extraction and analyzed by gas chromatography coupled with a mass spectrometer (GC-MS). Methylone was identified by a full scan mass spectral comparison to an analytical standard of methylone. For a definitive and conclusive confirmation and quantitation, methylone was also derivatized with heptafluorobutyric anhydride and analyzed by GC-MS. In all three fatalities, the deceased exhibited seizure-like activity and elevated body temperatures (103.9, 105.9 and 107°F) before death. Two of the three cases also exhibited metabolic acidosis. One of the three cases had prolonged treatment and hospitalization before death with symptoms similar to sympathomimetic toxicity, including metabolic acidosis, rhabdomyolysis, acute renal failure and disseminated intravascular coagulation. The laboratory results for this patient over the 24 h period of hospitalization were significant for increased lactate, liver transaminases, creatinine, myoglobin, creatine kinase and clotting times, and decreased pH, glucose and calcium. Peripheral blood methylone concentrations in the three fatal cases were 0.84, 3.3 and 0.56 mg/L. In conlusion, peripheral blood methylone concentrations in excess of 0.5 mg/L may result in death due to its toxic properties, which can include elevated body temperature and other sympathomimetic-like symptoms.

Quantitation of total 11-nor-9-carboxy-delta 9-tetrahydrocannabinol in urine and blood using gas chromatography-mass spectrometry (GC-MS).

Marijuana, which is made from crushing the leaves, flowers, and sometimes the stems of the plant Cannabis sativa, contains more than 30 cannabinoids. The major psychoactive cannabinoid is delta-9-tetrahydrocannabinol (THC). The major metabolite of THC, 11-nor-delta 9-carboxy-tetrahydrocannabionol (THC-COOH), is excreted in the urine primarily as a glucuronide conjugate and is commonly analyzed in biological specimens for detecting marijuana usage. The procedure described here involves the addition of deuterated internal standard THC-COOH-d9 into the sample followed by hydrolysis of conjugated THC-COOH by alkali. THC-COOH is extracted from urine or blood using liquid-liquid extraction followed by preparation of its trimethylsilyl derivatives. The analysis of derivatized THC-COOH is performed using gas-chromatography/mass spectrometry (GC/MS). Quantification of the drug in a sample is achieved by comparing the responses of the unknown sample to the responses of the calibrators using selected ion monitoring.

A fatality involving 1,3-propanediol and its implications in measurement of other glycols.

The decomposed body of a 45-year-old female was found, face down, in a mobile home, along with a suicide note and two antifreeze containers. Analysis of the body fluid collected from the decedent showed the presence of 58 mg/dL ethanol, but suspected ethylene glycol was not found in the sample. However, an unusually large peak of internal standard, 1,3-propanediol, was found in the sample. Gas chromatography-mass spectrometry analysis confirmed the presence of 1,3-propanediol in the sample. Using gas chromatography-flame-ionization detection, the concentration of 1,3-propanediol was determined to be 445 mg/dL. To our knowledge, this is the first report involving 1,3-propanediol as the cause of death. The study also highlights the importance for the close scrutiny of data, as 1,3-propanediol is a frequently used internal standard for the assay of glycols.

Using focus groups to develop a nutrition education video for high school students.

Focus group interviews were used to develop a nutrition education video and a teacher's guide for use in Florida high schools. Authors conducted a pilot and four focus group interviews with ninth grade students in five geographically distinct regions of Florida. Most students agreed that a video with scenarios or success stories would work well. Teens expressed interest in 10 topics: eating disorders; consequences of unhealthy eating; preparing quick, healthy meals; what constitutes a balanced diet; nutrition and fitness; weight control; food and the environment; the food guide pyramid; nutrition facts and fallacies; and food labels. Students suggested no more than three or four topics should be covered in the video, and the video cast should consist mainly of teen-agers with different body sizes, who were average, attractive, and from different ethnic backgrounds. Music was recommended only for transitions between scenes, as background, or during the credits.

Using focus group interviews to understand school meal choices.

Focus groups were used to examine adolescents' participation in school nutrition programs, their opinions about the meals served in these programs, and their suggestions for improving the meals. A pilot and four focus group interviews were conducted with ninth grade students in five geographically distinct regions of Florida. Male students and those from rural areas were more likely to participate in school meal programs than were females and those from suburban and urban areas. Students expressed intense dislike and distrust of school meals. School meals were criticized for taste, appearance, small serving sizes, and cost. Students overwhelmingly favored commercial food vendors serving lunch at school. Suggestions for improving school meals included improving the taste and appearance of food, offering a wider variety of foods, serving more fresh fruit and vegetables, lowering the price, increasing the serving sizes, teaching food service workers how to cook, and offering more condiments.