Hollow Fiber Liquid-Phase Microextraction At-Line Coupled to Capillary Electrophoresis for Direct Analysis of Human Body Fluids.

A simple and cheap all-in-one concept for at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to commercial capillary electrophoresis (CE) is demonstrated, which enables the direct analysis of complex samples. A disposable microextraction device compatible with injection systems of Agilent CE instruments is proposed, which consists of a short segment of a porous HF attached to a tapered polypropylene holder. The holder maintains a constant position of the HF in a CE vial during extraction and simultaneously guides the injection end of a separation capillary into the HF lumen for automated CE injection and analysis. In a typical analytical procedure, the HF is impregnated with a water-immiscible solvent, its lumen is filled with 5 µL of an aqueous acceptor solution, and the microextraction device is placed in a 2 mL glass CE vial containing 550 µL of a donor solution. The vial is agitated at 750 rpm for 10 min, and the resulting acceptor solution is injected directly from the HF lumen into the commercial CE. No additional manual handling is required, except for the transfer of the CE vial to the CE autosampler. Multiple complex samples can be simultaneously pretreated in a multiple-well plate format, thus significantly reducing the total analysis time. Suitability of the analytical method is demonstrated by the direct determination of model basic drugs (nortriptyline, haloperidol, loperamide, and papaverine) in physiological solutions, urine, and dried blood spot (DBS) samples. Repeatability of the method is better than 12.8% (%RSD), extraction recoveries range between 34 and 76%, and enrichment factors are 37-84. The method is linear in a range of 2 orders of magnitude (R2 ≥ 0.9977) with limits of detection of 0.7-1.55 µg/L. The method has a high potential for the direct analysis of DBS samples since DBS elution and HF-LPME are performed simultaneously during the 10 min agitation. The manual DBS handling is thus reduced to inserting the DBS punch into the CE vial only. Moreover, the universal character of the HF-LPME might extend the applicability of the method to a wide range of analytes/matrices, and combination with other commercial detectors might improve the selectivity/sensitivity of the CE analysis.

Nanogram/mL detection of nortriptyline: Preparation of a molecularly imprinted polymer for spectrophotometric determination of nortriptyline based on multivariate optimization methods.

In this work, the molecularly imprinted polymer was used as a selective sorbent in solid-phase extraction method for the spectrophotometric determination of nortriptyline at 239 nm. Molecularly imprinted polymer was synthesized by pyrrole as a functional monomer in the presence of nortriptyline as a template. Several factors, consist of the concentration of the monomer to template ratio, amount of initiator, stirring rate, reaction time, the pH of the buffer solution, amount of sorbent, loading time, shaking rate of loading, extraction time, and shaking rate of extraction were evaluated due to their effectiveness in the preparation and extraction capability of molecularly imprinted polymer. Multivariate optimization methods, such as Plackett-Burman and central composite designs, were employed to find and optimize the significant factors. Under the selected optimal conditions, molecularly imprinted polymer showed a linear range from 0.1 to 100 µmol/L (0.026 to 26 µg/mL) nortriptyline, a detection limit of 10.3 nmol/L (2.7 ng/mL), a highly repeatable (relative standard deviation of 3.7%) and reproducible response (relative standard deviation of 4.6%), and a good selectivity in the presence of structurally related molecules. Furthermore, molecularly imprinted polymer showed high extraction efficiency and was successfully used for the determination of nortriptyline in real samples.

Dispersive solid-liquid phase microextraction based on nanomagnetic Preyssler heteropolyacid: A novel method for the preconcentration of nortriptyline.

In this study, a new, simple, rapid, and efficient method combined with ultraviolet visible spectrophotometry and high-performance liquid chromatography analysis was developed for the extraction and determination of nortriptyline. The tendency of the Preyssler tungsten heteropolyacid, H14 [NaP5 W30 O110 ], immobilized on the surface of mesoporous nanomagnetite to adsorb the drug from the solution has been investigated. This method involves the use of an appropriate mixture of nanosorbent that was homogenized in disperser solvent (1.0 mL, ethanol). At first, the mixture containing the nanomagnetic sorbent and disperser solvent was injected into the aqueous sample, and a cloudy solution was formed. Subsequently, separation of the two phases was carried out using a magnet. In the second stage, analyte was desorbed from the sorbent by methanol as the optimal desorption solvent using sonication method. The elution solvent containing enriched analyte was introduced to the instruments for further analysis. Optimization of experimental conditions with respect to the extraction efficiency was investigated. The method was linear in the range of 25-5000, while the detection limit (LOD = 3SB /m) was 7.9 ng/mL and the limit of quantification (LOQ = 10SB /m) was 26.4 ng/mL. The relative standard deviation was 4.66%. The method was successfully applied to human hair samples.

[The determination of amitriptyline and its metabolite, nortriptyline, in the biological objects of the corpse by the high-performance liquid chromatography technique].

Tricyclic antidepressants are among the preparations that most frequently cause intoxication in adults and children; moreover, poisoning with these substances not infrequently has a fatal outcome. Medications belonging to this group, such as amitriptyline, are extensively used to manage manifestations of depression, anxiety, migraine, neuropathic pain, and hyperactivity syndrome. Amitriptyline overdosage causes non-specific symptoms of intoxication, and its clinical picture does not allow to identify the nature of a psychotropic xenobiotic. Of primary importance in connection with this is to establish the cause of intoxication or death by the clinical toxicological and forensic medical methods based on the results of the fast identification and quantitation of amitriptyline in biological materials including blood, urine, hepatic tissues, etc. The authors describe the method for the determination of amitriptyline and its principal physiological metabolite nortriptyline in biological objects with the help of high performance liquid chromatography (HPLC).

Managing the column equilibration time in hydrophilic interaction chromatography.

For a wide variety of hydrophilic interaction chromatography stationary phases, a repeatable partial equilibration was demonstrated in gradient elution after purging with as little as 12 column volumes of mobile phase. Relative standard deviations of retention time of on average ~0.15% could be obtained after 1 or 2 conditioning (blank) runs. The equilibration period must be kept strictly constant, otherwise selectivity changes occur, but this is not problematic on modern instruments. Partial equilibration was largely independent of stationary phase or gradient slope. Alternatively, full column equilibration is favoured for stationary phases that do not trap extensive water layers, and for materials with a wider pore size that have a lower surface area. Temperatures somewhat above ambient also shorten the equilibration time. Some stationary phases under optimum conditions can achieve full column equilibration using purging with ~12 column volumes, which is useful for rapid set-up of isocratic separations or for conventional gradient analysis.

Second-derivative synchronous fluorescence spectroscopy for the simultaneous determination of fluphenazine hydrochloride and nortriptyline hydrochloride in pharmaceutical preparations.

A rapid, simple, and highly sensitive second-derivative synchronous fluorimetric (SDSF) method has been developed for the simultaneous analysis of binary mixtures of fluphenazine hydrochloride (FLZ) and nortriptyline hydrochloride (NTP) in their co-formulated tablets. The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Deltalambda) = 120 nm in acetic acid. The different experimental parameters affecting the fluorescence intensity of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.25-3.0 and 1-10 microg/ml for FLZ and NTP respectively, with lower detection limits (LOD) of 0.05 and 0.18 microg/ml and quantitation limits of 0.15 and 0.53 microg/ml for FLZ and NTP respectively. The proposed method was successfully applied for the determination of the studied compounds in their synthetic mixtures and in commercial co-formulated tablets. The results obtained were in good agreement with those obtained by the reference methods.

A rapid and simple extraction of anti-depressant drugs by effervescent salt-assisted dispersive magnetic micro solid-phase extraction method using new adsorbent Fe3O4@SiO2@N3.

In this work, a rapid and simple method is applied using a newly synthesized nanoadsorbent for the extraction and preconcentration of the drugs Amitriptyline (AMT) and Nortriptyline (NRT), which are then determined using high performance liquid chromatography. We focus on the facile synthesis of Fe3O4@SiO2@N3, as a new and effective adsorbent, and the effervescent salt-assisted dispersive magnetic micro solid-phase extraction method. The applied method and modification of the surface of Fe3O4@SiO2 with the nitrogen-rich group lead to an increase in the interaction between the nanoadsorbent used and the analytes, and increase the extraction recovery. The accuracy of the synthesized nanoadsorbent is confirmed by the FT-IR, FE-SEM, XRD, and VSM analytical techniques. In order to obtain the best experimental conditions, optimization of the main variables involved is carried out using the central composite design method. Under the optimum experimental conditions, the limits of detection (LODs), linear dynamic ranges (LDRs), and relative standard deviations (RSDs for n = 5) for NRT and AMT were found to be as follow: LODs, 0.03 and 0.05 ng/mL; RSDs, 2.04 and 1.1 for NRT and AMT, respectively; and LDRs, 0.07-2000 ng/mL. Furthermore, the results obtained show that the nanoadsorbent can be used for five times with an extraction recovery more than 80% and 70% for AMT and NRT, respectively.

Liquid-phase microextraction for rapid AP-MALDI and quantitation of nortriptyline in biological matrices.

A liquid-phase microextraction (LPME) method using a micropipette with disposable tips was demonstrated for coupling to atmospheric pressure MALDI-MS (AP-MALDI/MS) as a concentrating probe for rapid analysis and quantitative determination of nortriptyline drug from biological matrices including human urine and human plasma. This technique was named as micropipette extraction (MPE). The best optimized parameters of MPE coupled to AP-MALDI/MS experiments were extraction solvent, toluene; extraction time, 5 min; sample agitation rate, 480 rpm; sample pH, 7; salt concentration, 30%; hole size of micropipette tips, 0.61 mm (id); and matrix concentration, 1000 ppm using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Three detection modes of AP-MALDI/MS analysis including full scan, selective ion monitor (SIM), and selective reaction monitor (SRM) of MS/MS were also compared for the MPE performance. The results clearly demonstrated that the MS/MS method provides a wider linear range and lower LODs but poor RSDs than the full scan and SIM methods. The LOD values for the MPE under SIM and MS/MS modes in water, urine, and plasma were 6.26, 47.5, and 94.9 nM, respectively. The enrichment factors (EFs) of this current approach were 36.5-43.0 fold in water. In addition, compared to single drop microextraction (SDME) and LPME using a dual gauge microsyringe with a hollow fiber (LPME-HF) technique, the LODs acquired by the MPE method under MS/MS modes were comparable to those of LPME-HF and SDME but it is more convenient than both methods. The advantages of this novel method are simple, easy to use, low cost, and no contamination between experiments since disposable tips were used for the micropipettes. The MPE has the potential to be widely used in the future because it only requires a simple micropipette to perform all extraction processes. We believe that this technique can be a powerful tool for MALDI/MS analysis of biological samples and clinical applications.

Suitability of 1-hexyl-3-methylimidazolium ionic liquids for the analysis of pharmaceutical formulations containing tricyclic antidepressants.

The reversed-phase chromatographic behaviour of six tricyclic antidepressants (amitryptiline, clomipramine, doxepin, imipramine, nortryptiline and maprotiline) was examined in this work with acetonitrile-water mobile phases, in the absence and presence of the ionic liquids 1-hexyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium tetrafluoroborate, which have interesting features for the separation of basic compounds, in terms of peak shape combined with reduced retention. Tricyclic antidepressants are low polarity drugs that strongly associate to the alkyl chains of conventional stationary phases. They are also positively charged in the usual working pH range (2-8) in reversed-phase liquid chromatography, due to their strong basic character. In consequence, they may interact with the residual ionised silanols present in conventional silica-based stationary phases, which is translated in stronger retention, and tailed and broad peaks. A simple chromatographic procedure for the control of tricyclic antidepressants in pharmaceutical formulations was developed using a C8 column and a mobile phase containing 30% acetonitrile/10 mM 1-hexyl-3-methylimidazolium chloride at pH 3, with UV detection. Intra- and inter-day precisions were usually below +1.0%, and intra- and inter-day bias (trueness) ranged between ‒2.1% and +2.4%, and between ‒3.0% and +2.3%, respectively. Sample preparation was simple and only required solubilisation and filtration previous to injection.

Novel method for the fast determination of ultra trace amount of nortriptyline in its pharmaceutical formulations by fast fourier transform continuous cyclic voltammetric technique at Au microelectrode in flowing solutions.

In this work a novel method for the determination of nortriptyline in flow-injection systems has been developed. The proposed method was used for the fast determination of nortriptyline in its pharmaceutical formulations. The developed technique is very simple, precise, accurate, time saving, and economical, compared to all of the previously reported methods. The effects of various parameters on the sensitivity of the method were investigated. The best performance obtained at pH value of 2, scan rate value of 30 V/s, accumulation potential of 400 mV, and accumulation time of 0.5 s. The proposed method has some advantages over other reported methods such as, no need for the removal of oxygen from the test solution, a subnanomolar detection limit, and finally the method is sufficiently fast for the determination of any such compound, in a wide variety of chromatographic methods. The potential waveform, consisting of the potential steps for cleaning, accumulation and potential ramp of analyte, was continuously applied on an Au disk microelectrode (12.5 microm in radius). The detection limit of the method was 2.0 x 10(-11) M. The relative standard deviation of the method at 1.2 x 10(-8) M was 2.1% for eight runs.

An estimate of the proportion of drug-facilitation of sexual assault in four U.S. localities.

In recent years, drugs including flunitrazepam, gamma-hydroxybutyrate, ketamine, and ethanol, have become popularly associated with drug-facilitated sexual assault. Other drugs are also candidates as factors in "drug facilitated sexual assault" (DFSA). The true extent of DFSA is not known, and is difficult to estimate. We recruited sexual assault complainants at four clinics in different parts of the U.S. to anonymously provide urine and hair specimens, and to answer questions about suspected drugging, drug use, and the sexual assault incident. Urine and hair specimens were tested for 45 drugs, including ethanol, and those pharmacologically capable of inducing sedation, amnesia, or impairment of judgment. Analytical test results were used to estimate the proportion of subjects, and the proportion of all complainants to the clinic in the same time period, who were victims of DFSA. Overall, cases of 43% of 144 subjects, and 7% of 859 complainants, were characterized as DFSA. Subjects underreported their use of drugs. The role of toxicological results and history in characterizing DFSA cases is discussed.

Injections from sub-µL sample volumes in commercial capillary electrophoresis.

A simple sample injection procedure compatible with commercial capillary electrophoresis (CE) instrumentation was developed, which enables handling sample volumes as little as 250nL for analytical applications where sample volume availability is of concern. Single-use micro-sampling inserts were prepared by thermal modification of polypropylene micropipette tips and the inserts were accommodated in standard CE vials in CE autosampler carousel. To ensure direct contact of separation capillary injection end with sample solution and to avoid possible damage to the capillary, a soft compression spring was placed at the bottom of the vial underneath the micro-sampling insert. Injections from sub-µL samples were carried out in conventional as well as in short-end injection mode, were compatible with standard i.d./o.d. (25-100µm/365µm) fused silica capillaries and with various background electrolyte solutions and detection modes. Excellent repeatability of replicate injections from 250nL to 3µL was achieved based on RSD values of quantitative analytical measures (peak heights ≤2.4% and peak areas ≤3.7%) for CE-UV-vis, CE-ESI-MS and CE-contactless conductivity detection of model basic drugs. The achieved RSD values were comparable with those for replicate injections of the drugs from standard CE vials. The reported concept of injections from micro-sampling inserts was further demonstrated useful in evaluation of micro-electromembrane extraction (µ-EME) of model basic drugs. Sub-µL volumes of operational solutions resulted in reduced lengths of µ-EME phases and improved extraction recoveries (66-91%) were achieved.

Postmortem distribution of tramadol, amitriptyline, and their metabolites in a suicidal overdose.

A case report involving a 34-year-old white male who was found dead at home by his roommate is presented. At the time of his death, he was being treated with tramadol/acetaminophen, metaxalone, oxycodone, and amitriptyline. The decedent's mother stated that he had been taking increasing amounts of pain medication in order to sleep at night. There were no significant findings at autopsy; however, toxicology results supported a cause and manner of death resulting from suicidal mixed tramadol and amitriptyline toxicity. This case reports the tissue and fluid distribution of tramadol, amitriptyline, and their metabolites in an acutely fatal ingestion in an effort to document concentrations of these analytes in 12 matrices with respect to one another to assist toxicologists in difficult interpretations.

Immunoassay reagents for psychoactive drugs. I. The method for the development of antibodies specific to amitriptyline and nortriptyline.

Amitriptyline and nortriptyline were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin (BSA). Rabbits inoculated with these conjugates yielded polyclonal antisera with high selectivity and good titers. This approach required novel spacer arms and new conjugation methods. The antisera produced were characterized with respect to their cross-reactivity with amitriptyline, nortriptyline and their hydroxy metabolites as well as selected structurally related compounds.

Breastfeeding and sertraline: a 24-hour analysis.

BACKGROUND: Few reports exist on the levels of antidepressants in breast milk or on observed behavioral effects, if any, of neonates who are breast-fed. Thus, a dilemma exists for women who would like to breast-feed but require psychotropic medications. METHOD: Analysis of sertraline levels was performed on eight samples of breast milk obtained over a 24-hour period, after 3 weeks of breastfeeding, from a lactating patient taking sertraline and nortriptyline. During this same 24-hour period, two serum samples each were taken from mother and child for analysis of sertraline and nortriptyline levels. After 7 weeks of exclusive breastfeeding, an additional serum sample was obtained from mother and child for analysis of sertraline levels. Drug metabolites were not measured. RESULTS: Breast milk levels of sertraline were lowest 1 hour before the ingestion of sertraline and highest 5 to 9 hours after ingestion of the drug. The infant's serum sertraline and nortriptyline levels were nondetectable. CONCLUSION: These data indicate that sertraline levels in breast milk vary substantially over 24 hours and appear to be lowest within the 2 hours before and 1 hour after ingestion of the medication, with the peak probably occurring between Hours 1 and 9 postingestion. However, the absence of detectable serum sertraline and nortriptyline levels in the infant suggests that if either medication is present in infant serum, its concentration would be extremely low. No abnormal occurrences have been noted in the development of the infant. It would be important in future studies to measure metabolites in addition to medication levels since the former have been associated with untoward events in an infant.

Infrared immunoassay.

An immunoassay method based on the labelling of an antigen with a transition-metal carbonyl organometallic marker and detection of the label by Fourier transform infrared (FT-IR) spectroscopy is described. The viability of this novel non-isotopic approach, termed infrared immunoassay (IRIA), has been evaluated in the quantitative determination of the tricyclic antidepressant nortriptyline.

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column.

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.

Radioimmunoassay of amitriptyline and nortriptyline in body fluids.

A radioimmunoassay for the measurement of amitriptyline and nortriptyline in serum, saliva, and urine is described. The sensitivity of the assay is such that 0.5 ng/ml of drug can be measured, although the assay does not distinguish between amitriptyline and nortriptyline. Other tricyclic compounds cross-react with the antiserum to varying degrees, but the metabolites of amitriptyline did not significantly cross-react. Total tricyclic compounds were detected in serum and saliva after single oral doses of amitriptyline, although the absorption was slow.

Spectrophotometric determination of some antidepressant drugs using 3-methylbenzothiazolin-2-one hydrazone.

A sensitive spectrophotometric method for the determination of amitriptyline hydrochloride, nortriptyline hydrochloride and doxepin hydrochloride in pure and dosage forms, is described. The method is based on the oxidative coupling of the drugs with 3-methylbenzothiazolin-2-one hydrazone in the presence of iron(III) chloride in 1 M hydrochloric acid. The commonly encountered excipients and additives do not interfere with the determinations. Results of the present method are comparable with those of official methods. The new method offers the advantage of simplicity and rapidity.