Determination of citalopram in fish brain tissue: benefits of coupling laser diode thermal desorption with low- and high-resolution mass spectrometry.

Recent state-of-the-art methods developed for the analysis of polar xenobiotics from different types of biological matrices usually employ liquid chromatography with mass spectrometry. However, there are limitations when a small amount of sample mass is available. For example, individual benthic invertebrates or fish tissue samples often weigh less than 100 mg (e.g., brain, liver) but are necessary to understand environmental fate and bioaccumulation dynamics. We developed ultra-fast methods based on a direct sample introduction technique. This included coupling laser diode thermal desorption with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). We then quantitated a common selective serotonin reuptake inhibitor (citalopram) in brain tissues of individual juvenile fish after in vivo exposure to environmentally relevant concentration. Two mass spectrometric methods based on low (LDTD-APCI-triple quadrupole (QqQ)-MS/MS) and high (LDTD-APCI-high-resolution product scan (HRPS)) resolutions were developed and evaluated. Individual instrument conditions were optimized to achieve an accurate and robust analytical method with minimum sample preparation requirements. We achieved very good recovery (97-108%) across the range of 1-100 ng g-1 for LDTD-APCI-HRPS. LDTD-APCI-QqQ-MS/MS showed poorer performance due to interferences from the matrix at the lowest concentration level. LDTD-APCI ionization was successfully validated for analysis of non-filtered sample extracts. Evaluation of final methods was performed for a set of real fish brain samples, including comparison of LDTD-APCI-HRPS with a previously validated LC-heated electrospray ionization-HRPS method. This new LDTD-APCI-HRPS method avoids the chromatographic step and provides important benefits such as analysis of limited sample masses, lower total sample volume (typically µL), and reduction in analysis time per sample run to a few seconds. Graphical abstract.

Analytical methodologies for the enantiodetermination of citalopram and its metabolites.

Citalopram (CIT) is a highly selective serotonin reuptake inhibitor (SSRI) frequently used in the treatment of major depressive disorders. It has a chiral centre in its structure and is used in therapy both as a racemic mixture (R,S-CIT) and a pure enantiomer (S-CIT). The differences between the pharmacokinetic and pharmacological profiles of the two enantiomers are well established. Consequently, the development of new efficient chiral analysis methods for their enantiomeric separation is a topic of great actuality. CIT metabolism is stereoselective as it is metabolized in chiral active metabolites, which retain considerable SSRI activity and contribute to the pharmacological effect. Chiral analytical methods are employed for the determination of enantiomeric ratio in pharmaceutical preparations and for monitoring the enantiomer levels in biological samples for therapeutic and toxicologic purposes. The current study reviews the published literature for the chiral analysis of CIT and its metabolites based on chromatographic and electrophoretic methods coupled with UV, fluorescence and mass spectrometry detectors.

Development of HPLC method coupled with fluorescence detection for simultaneous determination of donepezil HCl and trazodone HCl in spiked human plasma and tablets dosage forms.

OBJECTIVES: Elderly people with dementia are commonly suffered from sleep disorders. So, the use of Donepezil hydrochloride as anti-Alzheimer drug and Trazodone hydrochloride as antidepressants with hypnotic action is very important in these cases. This study reports about novel and sensitive RP-HPLC method with fluorescence detection for simultaneous bioanalytical determination of Donepezil hydrochloride (DON) and co-administered, Trazodone hydrochloride (TRA) in their pure forms, spiked human plasma and tablets. MATERIALS AND METHODS: Elution of both drugs was achieved with excellent resolution using a RP-C18 Hypersil Gold column and an isocratic mobile phase consisting of phosphate buffer (50mm, pH 4.6): methanol: acetonitrile (60:35:5) with a flow rate of 1.5mL/min and 20µL as injection volume. A Fluorescence detector at 300nm for excitation and 400nm for emission was used. RESULTS: Retention times were 4.3 and 6.3min for Donepezil hydrochloride and Trazodone hydrochloride, respectively. Linearity ranges of the assay were 25-1000 and 50-5000ng/mL and the limits of detection (LOD) and quantitation (LOQ) were 8.52, 15.47 and 25.81, 46.89ng/mL for Donepezil hydrochloride and Trazodone hydrochloride, respectively. CONCLUSION: The high sensitivity of the proposed method enabled the successful determination of the cited drugs in spiked human plasma with mean percentage of recoveries of 91.58±3.34 and 100.30±5.11 for Donepezil hydrochloride and Trazodone hydrochloride, respectively.

Development of bioanalytical HPLC method for simultaneous determination of the antialzhiemer, donepezil hydrochloride and the antidepressant, citalopram hydrobromide in raw materials, spiked human plasma and tablets dosage form.

OBJECTIVES: A novel, fast and sensitive HPLC method has been developed for the simultaneous bioanalytical determination of Donepezil hydrochloride (DON) and Citalopram hydrobromide (CTP) in raw materials, spiked human plasma and tablets. MATERIALS AND METHODS: Elution of both drugs was achieved with very good resolution using a RP-C18 chromatographic column, samples were analyzed using Hypersil Gold (100mm×4.6mm), 5µm particle size column and an isocratic binary mobile phase consists of phosphate buffer (0.05 M): acetonitrile (65:35). A Diode array detector at wavelength 232nm was used. Chromatographic separation was within a short run time (less than 7minutes) for both drugs. RESULTS: Retention times for DON and CTP were 4.5 and 5.8min, respectively. Linear calibration curves were obtained for DON and CTP over the concentration ranges of 0.1-10 and 0.1-50µg/mL. The mean extraction recoveries from spiked plasma were 93.22 and 92.64 for DON and CTP, respectively. The limits of detection and quantification were 0.017, 0.035µg/mL and 0.052, 0.106µg/mL for DON and CTP, respectively. CONCLUSION: The proposed method was successfully applied to the analysis of the cited drugs in raw materials, spiked human plasma and tablets with excellent accuracy and precision.

Determination of fluoxetine in Dermestes maculatus (Coleoptera: Dermestidae) by a spectrophotometric method.

The aims of this study were to detect and quantify fluoxetine, an antidepressant, from entomological samples. Larvae, pupae and adults of Dermestes maculatus (Coleoptera, Dermestidae) were reared on pig muscle previously treated with fluoxetine. The concentration selected, 2000mg/kg, emulates a fluoxetine overdose lethal to humans and laboratory animals. Thirty larvae on the fourth and fifth stages, 50 adults and several exuviae were analyzed for fluoxetine content. Detection of fluoxetine was performed by UV spectrophotometry at 270 and 277nm. All developmental stages of D. maculatus and exuviae were positive for fluoxetine. We also quantified the drug and no significant differences were found either between the days or the stages in the general model, but at 277nm a tendency of the concentration to decrease with time was observed. Concentrations of fluoxetine at 277nm were almost equal or greater than those at 270nm. This is the first study to detect and quantify fluoxetine from entomological samples and, in particular, from D. maculatus beetles.

Magnetized silane-coupling agent KH-570 based solid-phase extraction followed by gas chromatography-flame ionization detection to determine venlafaxine in human hair and aqueous environmental samples.

In the present study, a novel adsorbent, Fe3O4 magnetic nanoparticles (MNPs) functionalized by silane-coupling agent KH-570, was successfully synthesized. The prepared MNPs were characterized by Fourier-transform infrared spectroscopy and transmission electron microscopy. It was applied as functionalized magnetic nano-adsorbent for magnetic solid-phase extraction of trace levels of venlafaxine using gas chromatography with flame ionization detector. This method was developed and optimized for use in analysis of venlafaxine in human hair and aqueous environmental samples. The main factors influencing the extraction efficiency including pH of sample, amount of the MNPs, adsorption time, volume of sample, and desorption conditions such as volume of solvent and desorption time were studied and optimized. Under the optimized experimental conditions, good linearity was observed in the range of 1-1,000 µg L(-1) for aqueous environmental samples with correlation coefficients (R (2)) 0.996. The limits of detection and quantification were 0.1 and 0.5 µg L(-1), respectively. Good reproducibility with the relative standard deviations (n = 5) 3.21 % was obtained. The developed method was successfully applied to the extraction of venlafaxine from spiked human hair, river water, and surface water samples and the relative recoveries of 89.36, 93.43, and 94.99 % were obtained, respectively. The results indicated that Fe3O4/KH-570 MNPs have a satisfying extraction efficiency and can be served as a sensitive, inexpensive, and reliable method for analysis of antidepressant drugs such as venlafaxine in biological and aqueous environmental samples.

New validated HPLC methodology for the determination of (-)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase.

A new chromatographic method for the enantioseparation and the determination of (-)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157-164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (-)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets.

A validated silver-nanoparticle-enhanced chemiluminescence method for the determination of citalopram in pharmaceutical preparations and human plasma.

A simple and sensitive chemiluminescence (CL) method was developed for the determination of citalopram in pharmaceutical preparations and human plasma. The method is based on the enhancement of the weak CL signal of the luminol-H2 O2 system. It was found that the CL signal arising from the reaction between alkaline luminol and H2 O2 was greatly increased by the addition of silver nanoparticles in the presence of citalopram. Prepared silver nanoparticles (AgNPs) were characterized by UV-visible spectroscopy and transmission electron microscopy (TEM). Various experimental parameters affecting CL intensity were studied and optimized for the determination of citalopram. Under optimized experimental conditions, CL intensity was found to be proportional to the concentration of citalopram in the range 40-2500 ng/mL, with a correlation coefficient of 0.9997. The limit of detection (LOD) and limit of quantification (LOQ) of the devised method were 3.78 and 12.62 ng/mL, respectively. Furthermore, the developed method was found to have excellent reproducibility with a relative standard deviation (RSD) of 3.65% (n = 7). Potential interference by common excipients was also studied. The method was validated statistically using recovery studies and was successfully applied to the determination of citalopram in the pure form, in pharmaceutical preparations and in spiked human plasma samples. Percentage recoveries were found to range from 97.71 to 101.99% for the pure form, from 97.84 to 102.78% for pharmaceutical preparations and from 95.65 to 100.35% for spiked human plasma.

[Extraction techniques for analysis of venlafaxine and its metabolites in biological matrices]. / Techniki ekstrakcyjne w analizie wenlafaksyny i jej metabolitów w materiale biologicznym.

Venlafaxine (VEN), which was introduced into therapy in 1990s is one of the most often used antidepressants. The monitoring of its concentration in the organism is recommended, particularly in the case when a patient suffers of others illnesses and is treated with different drugs, which can interfere with VEN. The most popular diagnostic material for the determination of VEN level is blood. The present study is the review of actual reports on the methods of extraction of VEN and its metabolite from blood and other human diagnostic materials, like saliva and urine, and also from animals tissues. The paper shows the classic extraction methods, such as liquid-liquid extraction and solid-phase extraction. It also contains the modifications of these methods such as liquid-phase microextraction and cloud point extraction. According to the literature it can be stated that the best recovery of VEN and its main metabolite, O-demethylvenlafaxine, was obtained when the liquid-liquid extraction was used. The new, modified methods of extraction, are cost-effective, owing to the reduced use of solvents and also smaller volume of diagnostic material, but the results of the analysis, especially the recovery of the analytes, were lower than those obtained by classic methods of extraction.

Micellar electrokinetic chromatographic method for mianserin hydrochloride and analysis of degradation products by mass spectrometry.

A simple, stability-indicating micellar electrokinetic chromatography (MEKC) method was developed and validated for the analysis of mianserin hydrochloride in coated tablets. The method employed (hydroxymethyl)aminomethane (TRIS) 50 mM to which sodium dodecyl sulfate (SDS) 50 mM was added at pH 10.6 as the electrolyte and the voltage applied was 25 kV. The capillary used was 48.5 cm long (40.0 cm effective length and 50.0 µm i.d.) and the detection wavelength was 220 nm. Tetracycline was used as internal standard. The method was validated in accordance with the International Conference on Harmonization (ICH) requirements, which involved specificity, linearity, precision, accuracy and robustness. The stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using photodiode array detection. The degradation products formed under photolytic and oxidative conditions were investigated by electrospray ionization mass spectrometry. The method was linear over the concentration range of 50-130 µg/mL. The method was precise as demonstrated by an inter-day and intra-day relative standard deviation of less than 2.0%. The proposed validated MEKC method showed recoveries between 98.16 and 102.80% of the nominal contents. The Plackett-Burman design was applied for the robustness test in order to examine potential sources of variability by screening a large number of factors in a relatively small number of experiments.

Photodegradation kinetics, cytotoxicity assay and determination by stability-indicating HPLC method of mianserin hydrochloride.

A stability-indicating HPLC method for the determination of mianserin hydrochloride in coated tablets was developed and validated. Also, drug photodegradation kinetics and cytotoxicity were determined. Chromatographic analyses were performed in an Ace RP-18 octadecyl silane column (250 mm x 4.6 mm i.d., particle size 5 microm) maintained at ambient temperature (25 degrees C). The mobile phase was composed of methanol, 50 mM monobasic potassium phosphate buffer and 0.3% triethylamine solution adjusted to pH 7.0 with phosphoric acid 10% (85:15, v/v) in isocratic mode at a flow rate of 1.0 mL x min(-1). The performed degradation conditions were: acid and basic media with HCl 1.0 M and NaOH 1.0 M, respectively, oxidation with H2O2 3% and the exposure to UV-C light. No interference in the mianserin hydrochloride elution was verified by degradation products formed. Linearity was assessed and ANOVA showed non-significant linearity deviation (p > 0.05). Adequate results were obtained for repeatability, intermediate precision, accuracy and robustness. The photodegradation kinetics of mianserin hydrochloride was evaluated in methanol. The degradation of mianserin could be better described as zero order kinetic (r = 0.9982). The UV-C degraded samples of mianserin hydrochloride were also studied in order to determine the preliminary cytotoxicity in vitro against mononuclear cells.

Spectrofluorimetric determination of paroxetine HCl in pharmaceuticals via derivatization with 4-chloro-7- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl).

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the antidepressant paroxetine HCl (PXT) in its dosage forms. The method was based on coupling reaction of PXT with 4-chloro-7-nitrobenzo-2- oxa-1,3-diazole (NBD-Cl) in an alkaline medium (pH 8) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. The factors affecting the formation and stability of the reaction product were carefully studied and optimized. The fluorescence-concentration plot is rectilinear over the range 0.2-6 µg/mL with LOD of 0.08 µg/mL and LOQ of 0.24 µg/mL respectively. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. The mean percentage recoveries for paxetin and xandol tablets were 101.27 ± 1.79 and 101.33 ± 1.19 respectively. A proposal of the reaction pathway was postulated.

Analytical procedures for the determination of the selective serotonin reuptake inhibitor antidepressant citalopram and its metabolites.

The antidepressant citalopram (CIT) is a potent and highly selective serotonin reuptake inhibitor (SSRI) which has been introduced in therapy as a racemic drug. CIT has been used to treat central nervous system affective disorders such as depression, anxiety, obsessive-compulsive disorders, various phobias, borderline personality disorders, bipolar disorders as well as indications wherein inhibition of serotonin reuptake is desired. CIT is demethylated to demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT), which retain considerable activity as SSRIs. Therefore, in recent years, the monitoring of the levels of these analytes in biological fluids for toxicological and therapeutic purposes has been a target worthy of interest. In addition, the differences in activity between CIT enantiomers established the need to assess its behaviour in the field of pharmacological research. It is also necessary to develop analytical methodologies that make it possible to determine the levels of enantiomer concentrations. This review includes most of the published analytical methods for achiral assay of racemic CIT and its metabolites based on high-performance liquid chromatography coupled with UV, fluorescence and mass spectrometry detectors, capillary electrophoresis and gas chromatography with mass spectrometry detectors among others. With regard to the monitoring of enantiomers of CIT and of its metabolites, stereoselective methods based on chiral chromatographic columns, chiral additives in mobile phases and on the derivatization with a chiral reagent are also collected. In addition, different procedures of extraction are mentioned as well as liquid-liquid extraction, solid-phase extraction, solid-phase microextraction, automated online extraction or liquid-phase microextraction in different biological and environmental samples.

Development and validation of a HPLC-MS/MS method for the determination of venlafaxine enantiomers and application to a pharmacokinetic study in healthy Chinese volunteers.

An HPLC-MS/MS method has been developed and validated for the determination of venlafaxine enantiomers in human plasma and applied to a pharmacokinetic study in healthy Chinese volunteers. The method was carried out on a vancomycin chiral column (5 µm, 250 × 4.6 mm) maintained at 25°C. The mobile phase was methanol-water containing 30 mmol/L ammonium acetate, pH 3.3 adjusted with aqueous ammonia (8:92, v/v) at the flow rate 1.0 mL/min. A tandem mass spectrometer with an electrospray interface was operated in the multiple reaction monitoring mode to detect the selected ions pair at m/z 278.0 → 120.8 for venlafaxine enantiomers and m/z 294.8 → 266.7 for estazolanm (internal standard). The method was linear in the concentration range of 0.28-423.0 ng/mL. The lower limit of quantification was 0.28 ng/mL. The intra-and inter-day relative standard deviations were less than 9.7%. The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine enantiomers in 18 healthy volunteers. Validation parameters such as the specificity, linearity, precision, accuracy and stability were evaluated, giving results within the acceptable range. Pharmacokinetic parameters of the venlafaxine enantiomers were measured in the 18 healthy Chinese volunteers who received a single regimen with venlafaxine hydrochloride capsules. The results show that AUC((0-∞)) , C(max) and t(1/2) between S-venlafaxine and R-venlafaxine are significantly different (p < 0.05).

Quantitative determination of fluoxetine in human serum by high performance thin layer chromatography.

A high performance thin layer chromatographic method was developed and validated for the quantification of fluoxetine in human serum. Fluoxetine was extracted by liquid-liquid extraction method with diethyl ether as extraction solvent. Imipramine was used as internal standard. The chromatographic separation was achieved on precoated silica gel F 254 high performance thin layer chromatographic plates using a mixture of toluene/acetic acid glacial (4:5 v/v) as mobile phase. 4-Dimethylamino-azobenzene-4-sulphonyl chloride was used as derivatization reagent. Densitometric detection was done at 272 nm. The method was linear between 12.5 and 87.5 ng/spot, corresponding to 0.05 and 0.35 ng/microL of fluoxetine in human serum after extraction process and applying 25 microL to the chromatographic plates. The method correlation coefficient was 0.999. The intra-assay and inter-assay precisions, expressed as the RSD, were in the range of 0.70-2.01% (n=3) and 0.81-3.90% (n=9), respectively. The LOD was 0.23 ng, and the LOQ was 0.70 ng. The method proved be accurate, with a recovery between 94.75 and 98.95%, with a RSD not higher than 3.61% and was selective for the active principle tested. This method was successfully applied to quantify fluoxetine in patient serum samples. In conclusion, the method is useful for quantitative determination of fluoxetine in human serum.

[The level of fluoxetine in blood plasma and washed from eritrizite supernatant].

Antidepressant Fluoxetine and their major therapeutically active metabolite Norfluoxetine was monitored in albumene and glicoproteene of blood plasma and washed from eritrozite supernatant. In paper we describe rapid and reliable method using high-performance liquid chromatography for simultaneous measurement of plasma and supernatant concentration of Fluoxetine and Norfluoxetine. The described method has been successfully used in clinical and laboratory cases. Large interindividual variations in plasma and supernatan concentrations of Fluoxetine and their major metabolite have been shown. In albumene fractions of blood plasma the level of Fluoxetine is more higher then in supernatant. A successful development of the clinico-pharmacokinetic direction at the present stage is due to improvement and standardization of methods of clinical and pharmacokinetic examination which help to ensure a reliable pretreatment identification of patients potentially sensitive to pharmacotherapy with antidepressants.

Simultaneous determination of alprazolam and fluoxetine hydrochloride in tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography.

This paper describes validated HPLC and HPTLC methods for simultaneous determination of alprazolam (ALP) and fluoxetine hydrochloride (FXT) in pure powder and formulation. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length, 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer pH 5.5 (45 + 55, v/v) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using acetone-toluene-ammonia (6.0 + 3.5 + 0.5, v/v/v) as the mobile phase. Quantification in the HPLC method was achieved with UV detection at 230 nm over the concentration range 4-14 microg/mL for both drugs, with mean recovery of 99.95 +/- 0.38 and 99.85 +/- 0.56% for ALP and FXT, respectively. Quantification in the HPTLC method was achieved with UV detection at 230 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recovery of 99.32 +/- 0.45 and 99.78 +/- 0.81% for ALP and FXT, respectively. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ALP and FXT in pure powder and formulations.

Detecting fluoxetine and norfluoxetine in wild bird tissues and feathers.

The contamination of the environment with human pharmaceuticals is widespread and demand for such products is mounting globally. Wild vertebrates may be at particular risk from any effects from pharmaceuticals, because of the evolutionary conservation of drug targets. However, exposure of wildlife to pharmaceuticals is poorly characterised, partly due to challenges associated with detecting rapidly metabolised compounds. As part of a wider study on the behavioural effects of fluoxetine (Prozac) on Eurasian starlings (Sturnus vulgaris), we investigated which avian samples are best suited for detecting exposure to fluoxetine in free-living birds. We analysed plasma, various tissues and tail feathers (grown both in the wild and in captivity during the dosing period) from fluoxetine-treated birds (dosed daily with 0.035 mg kg-1 bodyweight for 28 weeks), and liver tissue and tail feathers from sham-dosed birds. We detected fluoxetine in only two of twelve plasma samples from dosed birds. In dosed birds, median concentrations of free fluoxetine/norfluoxetine in tissues (two hour post-final dose) were: 111.2/67.6 ng g-1 in liver, 29.6/5.7 ng g-1 in kidney, 14.2/4.0 ng g-1 in lung, 15.1/1.6 ng g-1 in brain. We estimated that fluoxetine would remain detectable in liver and kidney approximately 4.5 times longer (90 h) than in brain (20h). In dosed birds, fluoxetine was detected in feathers regrown during the dosing period (median concentration = 11.4 ng g-1) at concentrations significantly higher than in regrown feathers from control birds. Fluoxetine residues were detected in wild-grown feathers (grown before the birds were brought into captivity) at concentrations up to 27.0 ng g-1, providing some evidence of likely exposure in the wild. Our results show liver and kidney can be used for detecting fluoxetine in avian carcasses and provide a first indication that feathers may be useful for assessing exposure to fluoxetine, and possibly other pharmaceuticals.

Development of a fluvoxamine detection system using a Quenchbody, a novel fluorescent biosensor.

The misuse of psychotropic drugs intended for medical treatment represents a recent worldwide public health concern. Quenchbody (Q-body) is a novel fluoroimmunosensor that can detect an antigen immediately without additional reagents or washing steps. Here, we describe creating Q-bodies for the detection of the antidepressant fluvoxamine (FLV) and determining optimal conditions to achieve the highest fluorescence intensity (FI). We prepared five Q-bodies with the fluorophore labeled at either the N- or C- terminus and with different linker lengths. Fluorescence was measurable within minutes, indicating the interaction of Q-bodies with FLV. The normalized FI (FI ratio) of the N-terminus labeled Q-body increased approximately 1.5-fold upon FLV addition; Q-bodies labeled at the C-terminus did not significantly increase FI. Among the fluorescence dyes used in this study, Rhodamine 6G labeled Q-body showed the best FI ratio. EC50 values of the N-terminus labeled Q-bodies were similar (23.2-224nM) regardless of linker length or labeling dye. We examined whether the Q-body could be applicable to serum matrix instead of phosphate-buffered saline. The intact serum interfered strongly with the Q-body fluorescence. However, the FI ratios of the Q-body for FLV-spiked serum filtrate, for which proteins were removed by filtration, showed a dose-dependency for detecting FLV levels. Deproteinization, which does not interfere with Q-body fluorescence measurements, is likely necessary to detect serum FLV with high sensitivity. This study demonstrates the potential of Q-body probes as a tool towards developing creative immunoassay applications.

Development, validation and transfer into a factory environment of a liquid chromatography tandem mass spectrometry assay for the highly neurotoxic impurity FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine) in paroxetine active pharmaceutical ingredient (API).

This work describes the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a highly toxic impurity, FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine), in paroxetine active pharmaceutical ingredient (API), followed by the subsequent validation of the methodology and transfer into a global production/quality control environment. The method was developed to achieve a detection limit of 10ppb mass fraction of FMTP in paroxetine API. An LC-MS/MS method was chosen because it provided the required sensitivity and selectivity with minimal sample preparation. This paper discusses the issues with transferring such complex methodology to a production environment. Linearity, repeatability and reproducibility of the method were demonstrated. This work shows that it is possible using the same approach that would be used for the transfer of any analytical method from R&D to a manufacturing environment.