Journal of Chromatography B (v.836, #1-2)

A rapid, selective and robust direct-injection LC/hybrid tandem MS method has been developed for simultaneous screening of more than 250 basic drugs in the supernatant of enzyme hydrolysed equine urine. Analytes, trapped using a short HLB® extraction column, are refocused and separated on a Sunfire® C18 analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Independent data acquisition (IDA) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. The decision on whether or not to report a sample as a positive result was based upon both the presence of a MRM response within the correct retention time range and a qualitative match between the EPI spectrum obtained and the corresponding reference standard. Ninety seven percent of the drugs targeted by this method met our detection criteria when spiked into urine at 100 ng/ml; 199 were found at 10 ng/ml, 83 at 1 ng/ml and 4 at 0.1 ng/ml.
Keywords: Drug testing; Direct injection; Tandem hybrid mass spectrometry; Differential LC–LC gradient;

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography by Frédérique Renault; Eric Chabrière; Jean-Pierre Andrieu; Bernard Dublet; Patrick Masson; Daniel Rochu (15-21).
Human plasma paraoxonase (PON1) is calcium-dependent enzyme that hydrolyses esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. Human phosphate binding protein (HPBP) was discovered as contaminant during crystallization trials of PON1. This observation and uncertainties for the real activities of PON1 led us to re-evaluate the purity of PON1 preparations. We developed a hydroxyapatite chromatography for the separation of both HDL-associated proteins. We confirmed that: (1) HPBP is strongly associated to PON1 in HDL, and generally both proteins are co-purified; (2) standard purification protocols of PON1 lead to impure enzyme; (3) hydroxyapatite chromatography allows the simultaneous purification of PON1 and HPBP.
Keywords: Apolipoproteins; Human phosphate binding protein; Hydroxyapatite; Organophosphates; Paraoxonase;

A liquid chromatography–mass spectrometry (LC–MS) method was developed to screen and confirm veterinary drug residues in raw shrimp meat. This method simultaneously monitors 18 drugs of different classes, including oxytetracycline (OTC), sulfonamides, quinolones, cationic dyes, and toltrazuril sulfone (TOLS). The homogenized shrimp meat is extracted with 5% trichloroacetic acid. The extract is further cleaned using polymer-based SPE. A 50 mm phenyl column separates the analytes, prior to analysis with an ion trap mass spectrometer interfaced with an atmospheric pressure chemical ionization source. This method is able to confirm oxytetracycline residues at 200 ng/g, toltrazuril sulfone at 50 ng/g, sulfaquinoxaline at 20 ng/g, and the other 15 drugs at 10 ng/g or lower levels. An estimate of the level of residues can also be made so that only confirmed samples above action levels will be sent for quantitation. The method is validated with both fortified and incurred samples, using multiple shrimp species as well. This multi-class method can provide a means to simultaneously monitor for a wide range of illegal drug residues in shrimp.
Keywords: Shrimp; Multi-class; Multi-residue; High throughput; Veterinary drug; Confirmatory; Sulfonamide; Quinolone; Fluoroquinolone; Cationic dye; Oxytetracycline; Toltrazuril sulfone;

FOXE1 gene mutation screening by multiplex PCR/DHPLC in CHARGE syndrome and syndromic and non-syndromic cleft palate by Mario Venza; Maria Visalli; Isabella Venza; Claudia Torino; Rita Saladino; Diana Teti (39-46).
Denaturing high-performance liquid chromatography (DHPLC) has established itself as one of the most powerful tools for DNA variation screening. FOXE1, a highly GC-rich gene involved in syndromic cleft palate, is under investigation in thyroid dysgenesis, nonsyndromic cleft palate and squamous cell carcinoma. A technique for fast and simultaneous detection of sequence variants in the entire coding region of the FOXEl gene based on multiplex PCR/DHPLC is presented here. Given its characteristics of high sensitivity and rapidity, the testing strategy developed by us appears to be a reliable approach for FOXE1 analysis in the screening of a large population at risk.
Keywords: FOXE1 gene; Multiplex PCR/DHPLC; Sequence variant analysis; Oral clefting;

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC–UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC–UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC–MS/MS method. For the HPLC–UV method, good linearity was observed in the range 0–5 μg ml−1, and in the range 0–1 μg ml−1 for the LC–MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml−1 for the HPLC–UV method and the LC–MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml−1 for amiodarone and desethylamiodarone, respectively. The LODs of the LC–MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml−1 for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml−1 for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC–UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC–MS/MS method showed its applicability for single dose pharmacokinetic studies.
Keywords: Amiodarone; Desethylamiodarone; High-performance liquid chromatography; Liquid chromatography/electrospray ionization tandem mass spectrometry; Quantification; Validation; Plasma; Urine;

Preparation of inorganic molecularly imprinted polymers (IMIPs) with higher adsorption and selectivity has been developed on caffeine as model compound by sol–gel processes. In our study, by introducing pore-forming agent, lactic acid, into sol–gel process, the porosity of IMIPs was enhanced and the performance of IMIPs was thus improved. And, by introducing base catalyst in the sol–gel process, large pore volume was obtainable, and the caffeine adsorption of IMIPs was increased. Competition adsorption experiments between caffeine (CAF) and structure analogous molecule, theophylline (TH), were determined by HPLC analysis. It was found that adding pore-forming agent method produced better caffeine adsorption (ca. 20 μmol/g) than by adding base catalyst. But adding base catalyst method was found to yield better selectivity (ca. 4) (Selectivity (α) is the ratio of CAF bound to TH bound.) In addition, caffeine adsorption of IMIPs with template removed by calcination is two times that by extraction without sacrificing the selectivity of IMIPs.
Keywords: IMIPs; Inorganic molecularly imprinted polymers; Sol–gel; Template;

Resolution of common dietary sugars from probe sugars for test of intestinal permeability using capillary column gas chromatography by Ashkan Farhadi; Ali Keshavarzian; Jeremy Z. Fields; Maliha Sheikh; Ali Banan (63-68).
The most widely accepted method for the evaluation of intestinal barrier integrity is the measurement of the permeation of sugar probes following an oral test dose of sugars. The most-widely used sugar probes are sucrose, lactulose, mannitol and sucralose. Measuring these sugars using a sensitive gas chromatographic (GC) method, we noticed interference on the area of the lactulose and mannitol peaks.We tested different sugars to detect the possible makeup of these interferences and finally detected that the lactose interferes with lactulose peak and fructose interferes with mannitol peak. On further developing of our method, we were able to reasonably separate these peaks using different columns and condition for our assay. Sample preparation was rapid and simple and included adding internal standard sugars, derivitization and silylation. We used two chromatographic methods. In the first method we used Megabore column and had a run time of 34 min. This resulted in partial separation of the peaks. In the second method we used thin capillary column and was able to reasonably separate the lactose and lactulose peaks and the mannitol and fructose peaks with run time of 22 min.The sugar probes including mannitol, sucrose, lactulose, sucralose, fructose and lactose were detected precisely, without interference. The assay was linear between lactulose concentrations of 0.5 and 40 g/L (r 2  = 1.000, P  < 0.0001) and mannitol concentrations of 0.01 and 40 g/L (r 2  = 1.000). The sensitivity of this method remained high using new column and assay condition. The minimum detectable concentration calculated for both methods was 0.5 mg/L for lactulose and 1 mg/L for mannitol.This is the first report of interference of commonly used sugars with test of intestinal permeability. These sugars are found in most of fruits and dairy products and could easily interfere with the result of permeability tests. Our new GC assay of urine sugar probes permits the simultaneous quantitation of sucralose, sucrose, mannitol and lactulose, without interference with lactose and fructose. This assay is a rapid, simple, sensitive and reproducible method to accurately measure intestinal permeability.
Keywords: Capillary column gas chromatography; Lactulose; Lactose; Mannitol; Fructose; Intestinal permeability;

A novel method for quantitation of brain neurosteroid levels using HPLC with UV detection is described. In this simple and reliable method, testosterone from the brain and whole blood, and the internal standard, 17α-methyl testosterone, were extracted in 20% acetonitrile–phosphate buffer (pH 2.8), followed by solid phase extraction (SPE). The calibration curve was linear in concentration ranges from 0.1 to 10 ng from 0.2 g of tissue. We successfully applied this method to the analysis of endogenous testosterone in the male offspring of rats exposed to alcohol in utero. The concentration of testosterone at 21 post delivery in fetal alcohol exposure (FAE) group was significantly greater than the concentrations in either pair-fed or the ad libitum controls. These results support the usefulness of this method as a means of quantitating neurosteroids, and illustrate its applicability to fetal alcohol exposure.
Keywords: Endogenous testosterone; Fetal alcohol exposure; HPLC; UV;

Development and validation of a RP-HPLC method for quantification of isoflavone aglycones in hydrolyzed soy dry extracts by Isabela da Costa César; Fernão Castro Braga; Cristina Duarte Vianna Soares; Elzíria de Aguiar Nunan; Gerson Antônio Pianetti; Felipe Antonacci Condessa; Thiago Assis F. Barbosa; Ligia Maria Moreira Campos (74-78).
Isoflavones are widely used as an alternative treatment to hormone replacement therapy and also for prevention of several chronic diseases, including cancers. Genistein, daidzein and glycitein are the most abundant isoflavone aglycones found in soy extracts, where they also occur as glycosides. This paper describes the development and validation of an isocratic reversed-phase HPLC (RP-HPLC) method for the analysis of isoflavone aglycones, released after acid hydrolysis of soy dry extracts, used as pharmaceutical raw material. The quantification was carried out in a C18 endcapped column, using a mobile phase composed of 0.1% acetic acid and methanol (52:48), at a flow rate of 1.0 ml/min and diode array detection (DAD) at 254 nm. The method showed to be linear (r 2  > 0.99), precise (R.S.D. < 2%), accurate (recovery of 98.88% for daidzein and 98.12% for genistein), robust and specific.
Keywords: Isoflavones; Aglycones; Acid hydrolysis; RP-HPLC; Genistein; Daidzein; Glycitein;

A rapid and sensitive LC–MS/MS method for the quantification of ondansetron was developed and validated. The plasma samples were treated by a semi-automated liquid–liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Ondansetron and the internal standard (IS) granisetron were analyzed by combined reversed phase LC–MS/MS, with positive ion electrospray ionization, using multiple reactions monitoring (MRM). The statistical evaluation for this method reveals excellent linearity, accuracy and precision values for the range of concentrations 0.25–40.0 ng/mL. The proposed method enabled the reliable determination of ondansetron in bioequivalence studies after per os administration of a 4 or 8 mg tablet.
Keywords: Ondansetron; Liquid chromatography–tandem mass spectrometry; 96-well plates; Pharmacokinetic;

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin by Corey M. Ohnmacht; Shirley Chen; Zenghan Tong; David S. Hage (83-91).
Biointeraction studies based on high performance affinity chromatography were used to investigate the binding of human serum albumin (HSA) to two major phenytoin metabolites: 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). This was initially examined by conducting self-competition zonal elution experiments in which m-HPPH or p-HPPH were placed in both the mobile phase and injected sample. It was found that each metabolite had a single major binding site on HSA. Competitive zonal elution experiments using l-tryptophan, warfarin, digitoxin, and cis-clomiphene as site-selective probes indicated that m-HPPH and p-HPPH were interacting with the indole-benzodiazepine site of HSA. The estimated association equilibrium constants for m-HPPH and p-HPPH at this site were 3.2 (±1.2) × 103 and 5.7 (±0.7) × 103  M−1, respectively, at pH 7.4 and 37 °C. Use of these metabolites as competing agents for injections of phenytoin demonstrated that m-HPPH and p-HPPH had direct competition with this drug at the indole-benzodiazepine site. However, the use of phenytoin as a competing agent indicated that this drug had additional negative allosteric interactions on the binding of these metabolites to HSA. These results agreed with previous studies on the binding of phenytoin to HSA and its effects on the interactions of HSA with site-selective probes for the indole-benzodiazepine site.
Keywords: Phenytoin metabolites; Human serum albumin; High performance affinity chromatography; Biointeraction studies;

Fast simultaneous determination of urinary 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene by liquid chromatography–tandem mass spectrometry by Ruifang Fan; Yulian Dong; Wenbing Zhang; Yu Wang; Zhiqiang Yu; Guoying Sheng; Jiamo Fu (92-97).
A fast analysis method using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry was developed for the simultaneous determination of the 1-hydroxypyrene (1-OHP) and 3-hydroxybenzo[a]pyrene (3-OHBaP) in urine. Mass transitions were monitored at m/z 219.3–200.0 for 1-OHP and m/z 269.2–252.2 for 3-OHBaP. Only 10 min was needed for the analysis. The recovery was 60% for 3-OHBaP and 91% for 1-OHP, respectively. And the method detection limits were 0.49 μg/L for 1-OHP and 1.03 μg/L for 3-OHBaP. The inter- and intra-day relative standard deviations were in the range of 2.8–8.9% for 1-OHP and 9.7–20.8% for 3-OHBaP, respectively. The developed method was successfully used to measure urinary PAH metabolites of student volunteers in a high school.
Keywords: Polycyclic aromatic hydrocarbons; Metabolites; 1-Hydroxypyrene; 3-Hydroxybenzo[a]pyrene; Liquid chromatography–tandem mass spectrometry; Urine;

A validated method for the determination of sufentanil in human plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS) is described. Sufentanil was extracted from human plasma with solid-phase-extraction using deuterated sufentanil, [2H5]-sufentanil, as internal standard. Sufentanil and the internal standard were determined with an API 4000 tandem mass spectrometer equipped with a Turbo-V-Source operating in positive ESI mode on an Alltima HP HILIC straight phase column. The method showed a lower limit of quantification of 0.25 pg/ml (12.5 fg on column). The applicability of the method is shown in a clinical study, in which levels of sufentanil in plasma of parturients and arterial umbilical plasma of their neonates following patient-controlled epidural analgesia (PCEA) under several regimen treatments was analyzed.
Keywords: Sufentanil; PCEA; Chromatography; Tandem mass spectrometry; ESI; LC–MS/MS;

A selective HPLC method for determination of Huperzine A in Huperzia serrata Extract has been developed and validated. Huperzine A was dissolved in 0.01 mol/L HCl, chromatographed on an Agilent Zorbax SB-C18 (150 mm × 4.6 mm i.d., 5 μm) column, with a mobile phase consisting of methanol-1 mM l-Lysine water solution (50:50, v/v), and detected at 310 nm. The UV peak areas were used for quantitation of Huperzine A content. The correlation coefficient (R 2) of the calibration was 0.9999 over the range of 1–25 μg/ml and intra- and interday precision over this range were not more than 2%. The method was successfully applied to characterize and determine the Huperzine A in Huperzia serrata Extract.
Keywords: Huperzine A; HPLC;

We have recently seen the emergence of ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry as an alternative to traditional high-performance liquid chromatography techniques. The strengths of UPLC technology promote the ability to separate and identify drug compounds with significant gains in resolution and sensitivity and marked reductions in the overall time of analysis. As increased throughput is the desire of the practical toxicology laboratory, the aim of this study was to trial commercially available technology by assessment of the separation of several commonly encountered amphetamine-type substances. From injection of a poly-drug reference standard and whole blood extract, we successfully separated and identified amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine, MDA, MDMA, MDEA and ketamine in less than 3 min using the Acquity UPLC-Micromass Quattro Micro API MS instrumentation (Waters Corporation, USA). In addition to this significant reduction in overall run time, all peaks exhibited acceptable resolution using selected ion recording (SIR), with analysis indicating the capability to separate 5–11 peaks in 1.75 min using the current system parameters. From this introductory data, it is therefore indicated that the technological advancements defining ultra-performance liquid chromatography will allow it to serve as a powerful analytical tool for rapid throughput analysis.
Keywords: Ultra-performance liquid chromatography; UPLC; Amphetamine-type substances; Mass spectrometry; Methamphetamine; MDMA; Ketamine;

HPLC analysis of the second-generation antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma by Roberto Mandrioli; Maria Addolorata Saracino; Silvia Ferrari; Domenico Berardi; Ernst Kenndler; Maria Augusta Raggi (116-119).
A liquid chromatographic method with ultraviolet detection was developed for the analysis of the recent antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma. The analytes were separated on a C8 reversed phase column, using a mobile phase composed of acetonitrile and a 12.3 mM, pH 3.0 phosphate buffer containing 0.1% triethylamine (35:65, v/v). Clomipramine was used as the Internal Standard. Using a solid phase extraction procedure with C2 cartridges high extraction yields (>94%) and good purification from matrix interference were obtained. Good linearity was obtained in the 7.5–250.0 ng mL−1 range for sertraline and in the 10–500 ng mL−1 range for N-desmethylsertraline. The analytical method was validated in terms of precision, extraction yield and accuracy. These assays gave R.S.D.% values for precision always lower than 3.9% and mean accuracy higher than 90%. Thanks to its good selectivity, the method proved to be suitable for the analysis of plasma samples from patients treated with sertraline as either monotherapy or polypharmacy.
Keywords: Sertraline; N-desmethylsertraline; Human plasma; Liquid chromatography; Solid phase extraction;

Plasma measurements of levodopa and its major metabolites including dopamine and 3-O-methyldopa have been limited by cumbersome methods and poor sensitivity within relatively narrow ranges of plasma levels. We now report a modification of an HPLC method that permits concomitant measurements of a wide range of concentrations of levodopa, dopamine (DA), carbidopa, 3-O-methyldopa (3-OMD) and 3,4-dihydroxyphenyl acetic acid (DOPAC) from one HPLC injection. The recoveries ranged from 77 to 107% with an intra-day precision around 5% (CV) and inter-day CV's about 10–20%. This validated method will simplify pharmacokinetic studies of levodopa and its metabolites for mechanistic studies or therapeutic clinical monitoring which play a crucial role in development of strategies to prolong motor benefits from individual doses and reduce involuntary movements called dykinesias.
Keywords: Levodopa; Plasma measurements; HPLC; Carbidopa; 3-O-Methyldopa; Dopamine; DOPAC (3,4-dihydroxyphenyl acetic acid);

Doping control for metandienone using hair analyzed by gas chromatography–tandem mass spectrometry by Marie Bresson; Vincent Cirimele; Marion Villain; Pascal Kintz (124-128).
A sensitive, specific and reproducible method for the quantitative determination of the anabolic metandienone in human hair has been developed. The preparation involved a decontamination step with methylene chloride. The hair sample (about 50 mg) was solubilised in 1 ml 1 M NaOH, 10 min at 95 °C, in presence of 2 ng of nandrolone-d 3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase extraction (Isolute C18 eluted with methanol) and a liquid–liquid extraction with pentane. The residue was derivatized by adding 5 μl MSTFA/NH4I/2-mercaptoethanol (250 μl; 5 mg; 15 μl) and 45 μl MSTFA, then incubated for 20 min at 60 °C. A 1 μl aliquot of derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl–95% methylsiloxane, 30 m × 0.32 mm i.d., 0.25 μm film thickness) of a Hewlett Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Metandienone was identified using three transitions (its daughter ions at m/z 339 and 206 for the parent 444 and 191 for 206) using a Waters Quattro Micro MS–MS system. The transition m/z 444 to 206 has been used as quantification transition and the others as identification transitions. The assay was capable of detecting 2 pg/mg of metandienone when approximately 50 mg of hair material was processed. Linearity was observed for metandienone concentrations ranging from 2 to 500 pg/mg with a correlation coefficient of 0.9997. Intra-day and between-day precisions at 50 pg/mg were 13.4–16.5% and 22.0%, respectively, with an extraction recovery of 48%. The analysis of hair, cut into four segments, obtained from an athlete, revealed the presence of metandienone at the concentrations of 78, 7, 10 and 108 pg/mg in each segment of hair (0–1, 1–2, 2–3 and 3 cm to the tip).
Keywords: Metandienone; Doping; Hair; MS–MS;

Atrazine is an herbicide which has shown potential antimalarial effects both in vitro and in vivo in rats. In order to study the metabolism of atrazine in rat livers, we developed a sensitive LC/MS/MS method for the identification of atrazine and several of its metabolites. Using this method, we identified one previously unreported metabolite with a mass of 211 Da in addition to two known metabolites. This new metabolite was confirmed to be N-ethyl-6-methoxy-N′-(1-methylethyl)-1,3,5-triazine-2,4-diamine, also known as atraton, by comparison of the LC/MS/MS mass spectra and the retention time to those of a commercial standard.
Keywords: Atrazine; Atraton; LC/MS/MS; Metabolite;