Atomoxetine: A Review On Analytical Method Development And Validation For Quantification Of Bulk And Pharmaceutical Dosage Form By High Performance Liquid Chromatography

Abstract

Atomoxetine is the efficacious drug for treating attention deficit hyperactivity disorder (ADHD) in adults and children. This drugs clinical and pharmaceutical analysis necessitates efficient analytical techniques for quality assurance, pharmacodynamics, pharmacokinetic, and stability investigations. We have reviewed in detail the literature from many journals related to analytical and pharmaceutical chemistry, and we have looked at instrumental analytical methods that were created and used to find a drug in bulk drugs, formulations, and biological fluids, either by itself or in combination with other drugs. This review covers the most recent analytical methods including HPLC, HPTLC, RP HPLC and liquid chromatography were reported.

Keywords

Introduction

Analytical Method Validation

Validation is a method-based approach to ensure a method's suitability as a quality control tool for analytical measurements. Any analytical measurement's goal is to produce accurate, dependable, and consistent results. The use of validated analytical techniques is crucial to accomplishing this objective. An analytical method consists of techniques, methods, procedures, and protocols. Analytical method validation includes the determination of accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and robustness. The results from method validation can be used to moderate the quality, reliability, and consistency of analytical results, which is an integral part of any good analytical practice. The majority of laws and standards of quality governing laboratories also entail the validation of analytical procedures.

Analytical Methods for Atomoxetine

• IP-2022 (Atomoxetine capsules) - Liquid chromatography- Mobile phase: Buffer (pH 2.5): Acetonitrile (62:38), Column: 7.5 cm × 4.6 mm (3.5 µm), Flow rate: 1.5mL/min, Wavelength: 220 nm.[5]
• BP-2020 - Liquid chromatography- Mobile phase: Propanol R: Buffer (pH 2.5) (27:73 v/v), Column: 0.15 m × 4.6 mm (3.5 µm), Flow rate:1.0 mL/min, Detection: UV 215 nm.[6] 
• USP-2021 (Atomoxetine capsules) - Liquid chromatography- Mobile phase: Buffer (38:62), Column: 4.6 m × 7.5 cm; 3.5 µm packing L7, Flow rate: 1.5 mL/min, Detector: UV 220 nm.[7]

Reported Methods for Atomoxetine

Mullen JH et al

For the measurement of atomoxetine and its metabolites (4-hydroxyatomoxetine, N-des-methylatomoxetine, and 4-hydroxyatomoxetine-O-glucuronide), a sensitive and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) approach has been developed for human plasma and urine. The chromatographic system consisted of a Brownlee Spheri-5 C18 polyfunctional column (4.6mm × 100 mm; dp, 5µm) with the use of a binary gradient (mobile phase A: water, mobile phase B: 5mM ammonium acetate, 47.2mM formic acid, 4mM trifluoroacetic acid in acetonitrile-water (85:15, v/v)). The approach demonstrated accuracy and precision for the analytes in all species when using stable-labelled internal standards; inter-batch accuracy (%RE, percent relative error) was within 100 ± 13%, and inter-batch precision (%RSD, relative standard deviation) was within 11%. Plasma and urine (with or without the deconjugation reagent) were shown to be stable, as were the analytes in clean solutions and the reconstitution solvent. The technique proved straightforward, reliable (used to analyse several hundred clinical trial samples), and scalable to handle large volumes of data.[8]

Sellers JA et al

Atomoxetine hydrochloride has been the subject of the development and validation of a normal-phase isocratic chiral liquid chromatographic technique. The conditions also distinguish the phenyl des-methyl analog and the para and meta positional isomers of atomoxetine in addition to its S-enantiomer. Method development strategies included capillary electrophoresis using a single isomer of heptakis6-sulfato--cyclodextrin modifier,  the use of an octyl stationary phase with a sulfated--cyclodextrin mobile phase additive, and evaluation of polysaccharide-based chiral stationary phases with nonaqueous mobile phases. All three methods produced satisfactory results for atomoxetine isolation from related compounds; the latter two were retained as backups in case the former proved ineffective. The final technique conditions use a mobile phase of hexane/IPA/DEA/TFA (85/15/0.15/0.2, v/v/v/v) at 1.0 ml/min and a Chiralcel OD-H column.[9]

Zhu HJ et al

The first HPLC-fluorescence technique was created and approved to measure atomoxetine in human plasma. Under moderate circumstances, atomoxetine was derivatized using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl). It was then isocratically separated on a C18 column using an HPLC system with fluorescence detection (?ex: 318 nm, ?em: 448 nm). The separation was performed at room temperature using acetonitrile: water (75:25, v:v) with the flow rate set at 1.0 mL/min. Over the concentration range of 1–1000 ng/mL, a linear calibration curve (r = 0.999) was produced. With a S/N ratio of 3, the detection limit was 0.3 ng/mL. Within-day and inter-day variability had relative standard deviations of ?8.30% and 7.47%, respectively.[10]

Patel C et al

The detection of atomoxetine has been reported using high performance liquid chromatography (HPLC) with liquid scintillation counting (LSC) detection and costly liquid chromatography tandem mass spectrometry (LCMS). There is currently no technique described in the literature for atomoxetine determination utilizing HPLC and UV detection. In this study, we report on a novel HPLC technique that uses tertiary butyl methyl ether, liquid–liquid extraction, and a UV detector to determine the amount of atomoxetine. The HPLC system consisting of pump-PU-980, detector-UV-975 was used. Chromatographic separation was carried out at UV wavelength 272 nm, by using Agilent SB-C18 (4.6mm × 150 mm, 5µm) column. The mobile phase consisted of acetonitrile: 5mM heptane sulphonic acid buffer with 1% (v/v) of triethylamine, pH adjusted to 4.8 by glacial acetic acid (40:60, v/v). At 1 ml/min, the mobile phase flow rate was maintained constant. Over the concentration range of 0.05–3.0 µg/ml, this technique was proven to be linear. 0.05 µg/ml was the quantitative limit. The observed mean ± S.D. pharmacokinetic parameters Cmax, Tmax and AUC0-t were 0.40 ± 0.06 µg/ml, 3.40 ± 0.42 h and 1.34 ± 0.52 µg h/ml, respectively.[11]

Guo W et al

To quantify atomoxetine, a novel drug for attention deficit/hyperactivity disorder, in human plasma, an HPLC technique with UV detection (210 nm) was devised and validated. An isocratic mobile phase of acetonitrile: phosphate buffer (39:61, v/v, pH 6.6) on a reverse phase Inertsil C18 column was used to separate the analyte and internal standard (maprotiline) after a two-step liquid–liquid extraction with diethyl ether. In plasma, linearity was confirmed between 3.12 and 200 ng/mL of atomoxetine. 2.5 ng/mL is the lowest limit of detection (S/N = 10). Within- and between-batch precisions of 4.9–14.4% and 4.7–13.1%, respectively, were obtained during the validation of this HPLC technique. There were biases within the batch of ?1.9 to 1.4% and between the batches of 0.1–13.8%, respectively.[12]

Kamat SS et al

Atomoxetine HCl in capsules can now be assayed using a high-performance liquid chromatographic method (HPLC) that is quick, easy to use, and sensitive. Using a reversed phase C18 analytical column (150 × 4.6 mm i.d. 5 µm particle size) and a mobile phase made up of acetonitrile and monobasic potassium dihydrogen orthophosphate (95:5 v/v), the HPLC analysis was performed at 269 nm using UV detection. The assay's sensitivity, specificity, and repeatability for determining atomoxetine HCl in this dose form were demonstrated by the validation data. From 1 to 10 µg mL-1, the calibration curves were linear (R2 > 0.997). The method's accuracy ranged from 98.13 to 101.5%. Relative standard deviations (RSD) of the mean between and within assays were less than 1.0%.[13]

Gavin PF et al

The article describes the creation of an ion-pairing HPLC technique and related system suitability factors for the quality by design analysis of atomoxetine hydrochloride (LY139603 HCl). In order to optimize conditions and show method resilience for the separation of atomoxetine and contaminants, statistically planned experiments were employed. Potential method conditions were assessed for their capacity to satisfy design objectives. This peak pair's resolution serves as a test for the suitability of the system without requiring impurity reference standards. The separation of two early eluting impurities, phenyl methylaminopropanol (PMAP (±)3-methylamino-1-phenylpropanol) and mandelic acid, is correlated to the separation of other impurities that elute near the main sample component.[14]

Nagaraju V et al

Within the category of psychoanaleptics include atomoxetine, venlafaxine, and fluoxetine. Although atomoxetine is used to treat ADHD, the other two medications are used to treat depressive disorders. Pharmaceutical items can now be tested for atomoxetine, venlafaxine, and fluoxetine using an easy-to-use, quick, isocratic, high-performance liquid chromatographic (HPLC) technology. In the HPLC method, the mobile phase is acetonitrile-potassium dihydrogen phosphate buffer (0.05 M adjusted to pH 3.0 with phosphoric acid) (45:55 v/v), and the separation is performed by reversed phase HPLC on a Nova-Pak C18 column (15 cm 3.9 mm id). The flow rate is 0.8 mL/min at room temperature (25oC), with UV detection enabled at 226 nm. Limit of detection (LOD), limit of quantitation (LOQ), robustness, specificity, linearity, precision, and accuracy were all verified for the method.[15]

Kamat SS et al

Atomoxetine HCl in capsules can now be assayed using a high-performance thin layer chromatographic method (HPTLC) that is quick, easy to use, and sensitive. This study employed a mobile phase made up of a combination of acetonitrile and glacial acetic acid with UV detection at 269 nm and a normal phase (silica gel 60 F 254) as a stationary phase. The assay's sensitivity, specificity, and repeatability for determining atomoxetine HCl in this dose form were demonstrated by the validation data. From 100 to 1000 µg mL-1, the calibration curves were linear (R2 > 0.997). The method's accuracy ranged from 99.12 to 99.80%. Relative standard deviations (RSD) of the mean between and within assays were less than 2.0%. An exact and accurate analysis of atomoxetine HCl in its pharmaceutical forms was made possible by the suggested method.[16]

Patel SK et al

The development of a stability-indicating RP-HPLC method for atomoxetine hydrochloride (ATX) detection in the presence of its degradation products, which are produced via forced decomposition experiments, is presented in this study. The drug material was exposed to oxidation, base, acid, dry heat, and photodegradation stress conditions. The medication proved vulnerable to oxidation, basic, acid, and wet and dry heat deterioration in stability testing. Under the evaluated photolytic conditions, it was shown to be stable. On a Phenomenex C18 column (250 × 4.6 mm id, 5µm particle size), the medication was successfully separated from the breakdown products generated under stress conditions by employing acetonitrile-methanol-0.032 M ammonium acetate (55 + 05 + 40, v/v/v) as the mobile phase at 1.0 mL/min and 40oC. Following RP-HPLC over the concentration range of 0.5-5 µg/mL, photodiode array detection at 275 nm was utilized for quantification, with a mean recovery of 100.8 ± 0.4% for ATX. The procedure is particular, accurate, and reproducible for estimating ATX, according to statistical research. The technique can be utilized as a stability-indicating tool since it successfully isolates the medication from its degradation products.[17]

Dogrukol-Ak D et al

The quantification of atomoxetine in medications and human plasma has been reported using a liquid chromatographic technique. Following a straightforward, one-step protein precipitation with methanol, plasma samples were analyzed. Atomoxetine and carbamazepine (an internal standard) were separated by chromatography using the ideal mobile phase of a methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (35:15:50, v/v/v). The human plasma and the mobile phase respective limit of quantification values for atomoxetine were 45.2 and 49.5 ng/ml. Atomoxetine can be found in medicines and human plasma using a fully proven approach that is repeatable and selective.[18]

Prajapati HR et al

Atomoxetine hydrochloride in bulk and pharmaceutical formulation was determined using a reversed phase high performance liquid chromatographic (RP–HPLC) method that was developed and verified later. A PerkinElmer Brownlee analytical C8 column (260 mm × 4.6 mm, 5 µm) was used for the separation, and the eluent ratio was 80:20 v/v for methanol: 50 mM monopotassium phosphate buffer (pH adjusted to 6.8 with 0.1 M sodium hydroxide). UV detection was carried out with a flow rate of 1.0 mL/min at 270 nm. Atomoxetine hydrochloride was found to have a correlation coefficient of 0.997. The recovery fell between 99.94 to 100.98%, and 5.707 µg/mL was determined to be the limit of quantification.[19]

Marchei E et al

The measurement of atomoxetine (ATX) and its metabolites 4-hydroxyatomoxetine (4-OH-ATX) and N-des-methylatomoxetine (N-des-ATX) in plasma, urine, oral fluid, and sweat is reported using a system based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) with duloxetine serving as the internal standard. The analytes were extracted using two millilitre aliquots of tertbutyl methyl ether from 0.5 millilitre biological fluids and a sweat patch. In the mobile phase, the organic layer was redissolved after evaporating. With a reverse-phase column and an isocratic mobile phase composed of 40% water and 60% 5 mM ammonium acetate, 47.2 mM formic acid, and 4 mM trifluoroacetic acid in acetonitrile–water (85:15, v/v) at a flow rate of 0.5 mL/min, chromatographic separation was performed. The analytical limits for the three analytes were as follows: 0.5 ng/mL oral fluid and plasma, 10 ng/mL urine, and 1 ng/patch utilizing 0.5 mL biological fluids or one sweat-patch. Over the calibration ranges, the calibration curves had a linear relationship with r2 > 0.99. The mean recoveries in several biological matrices were consistently greater than 65% at three concentrations that covered the assay's linear dynamic range. Using standard and non-traditional biological matrices from patients undergoing medication treatment, this technique was used to therapeutically monitor ATX and its metabolites 4-OH-ATX and N-des-ATX.[20]

Choi CI et al

Atomoxetine, a strong and specific inhibitor of the presynaptic norepinephrine transporter used to treat attention deficit/hyperactivity disorder, is mostly metabolized into 4-hydroxyatomoxetine (4-HAT) and N-desmethylatomoxetine (N-DAT). 4-HAT has pharmacological action that is comparable to atomoxetine. A straightforward, quick, and sensitive liquid chromatography analytical technique using tandem mass spectrometry (LC–MS/MS) for determining 4-HAT and N-DAT in human plasma has been created and verified by us. Following methyl t-butyl ether liquid-liquid extraction, the analytes were separated by chromatography using a reversed-phase Luna C18 column (2.0 mm × 100 mm, 3 m particles) and a mobile phase consisting of 10 mM ammonium formate buffer (pH 3.5)–methanol (10:90, v/v). The analytes were quantified using MS/MS detection in ESI positive ion mode. The mobile phase flow rate was 250 µl/min, and the internal standard (IS, metoprolol), 4-HAT, and N-DAT retention durations were 0.9, 1.0 and 1.0 min, respectively. For 4-HAT and N-DAT, the calibration curves were linear over 0.05–20 ng/mL and 0.1–20 ng/mL, respectively. Using 200 µl of human plasma, the lowest quantification limits for 4-HAT and N-DAT were 0.05 and 0.1 ng/mL, respectively. For the intra- and inter-day validation of 4-HAT and N-DAT, the mean accuracy and precision were both within allowable bounds. When compared to previously published analytical methods, the LC-MS/MS method demonstrated better sensitivity for the quantification of the two primary metabolites of atomoxetine in human plasma. Human pharmacokinetic research was successfully conducted using the approved methodology.[21]

Ulu ST et al

Using a reversed phase separation, pre-column derivatization with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride), and fluorescence detection, a novel and sensitive approach was devised for the quantification of atomoxetine in human plasma and urine. Using methanol: water (85:15 v/v) as a mobile phase in an isocratic elution mode, liquid chromatography separation was accomplished on an Inertsil C18 column. A fluorescence detector tuned at 375 and 537 nm for excitation and emission wavelengths, respectively, was used to monitor the eluents. The internal standard utilized was mexiletine. Linearity, detection limit, quantification limit, precision, accuracy, recovery, robustness, and system appropriateness were all confirmed for the approach. For both urine and plasma, the concentration ranges covered by the technique were 25–3000 ngmL-1 and 10–2250 ngmL-1, respectively. Atomoxetine was recovered from plasma and urine with mean values of 97.39 and 96.77%, respectively.[22]

Papaseit E et al

One treated child's hair and five treated teenagers' hair were used to figure out the presence of ATX and its primary metabolites (4-hydroxyatomoxetine, or 4 hydroxy-ATX, and N-desmethylatomoxetine, or des-methyl-ATX), using a method centred around liquid chromatography–tandem mass spectrometry (LC–MS/MS). Hair samples were digested overnight at 45oC using 2 ml of 1 M NaOH after duloxetine was added as an internal standard. Then, taking two separate 2 ml aliquots of tert-butyl methyl ether, analytes were extracted from the alkaline solution. Using a reverse-phase column and a mobile phase of 40% water–60%, 5 mM ammonium acetate, 50 mM formic acid, and 4 mM trifluoroacetic acid in acetonitrile–water (85:15, v/v), chromatographic separation was performed at room temperature. Multiple reaction monitoring was used to run the mass spectrometer in positive ion mode. For all analytes under inquiry, the method's concentration range was linear over 0.2–50 ng/mg hair. The analytical recovery ranged from 33.1% to 76.1%, depending on the analyte under consideration, and both intra- and inter-assay precision and accuracy were consistently less than 20%. In hair samples, the concentrations of ATX and 4-hydroxyATX varied from 0.2 to 2.0 ng/mg of hair and from 0.3 to 1.0 ng/mg, respectively.[23]

Hills V et al

This study is determined by using a straightforward, quick, accurate, exact, and specific RP-HPLC approach in both bulk and prescription dosage forms. Chromatography was carried out at a flow rate of 1.2 mL/min using a mobile phase consisting of a mixture of phosphate buffer and acetonitrile (60:40 v/v) packed with 5µm particles on an Inertsil C18 column (150 x 4.6 mm). The atomoxetine was successfully eluted with a retention time of 3.59 minutes after UV detection at 270 nm. The analysis's findings were statistically verified for accuracy, precision, linearity, specificity, and robustness in accordance with ICH norms. Between 10 to 200 µg/mL, linear calibration plots were produced, and the methods percentage RSD was found to be less than 2%. The excipients in the formulations do not impede the assay process, and the recommended approach can be effectively used for the regular estimation of atomoxetine in solid dosage forms.[24]

Appel DI et al

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to establish a quick, easy, and accurate way to quantify atomoxetine. This test is the first to use electrospray ionization in LC-MS/MS measurement of atomoxetine. The internal standard was set at d3-atomoxetine, or deuterated atomoxetine. The sample preparation process involved the use of direct protein precipitation. Both human plasma and in vitro cellular samples were used to validate this methodology. For both human plasma and cellular samples, the lower limit of quantification was 3 ng/mL and 10 nm respectively. For both human plasma and cellular samples, the calibration curves were linear between 3 to 900 ng/mL and 10 nm to 10 µm, respectively (r2 > 0.999). Quality control samples in both human plasma and cellular lysate were used at three different concentrations to assess the assay's accuracy and precision both within and between days. Additionally, satisfactorily proven were sample run stability, assay selectivity, matrix influence, and recovery. The sensitivity or ease of sample preparation of this assay is superior to that of previously described LC-MS and LC-MS/MS procedures. This assay can be used to analyse atomoxetine in in vitro cellular samples as well as human plasma.[25]

Prajapati HR et al

A novel high-performance thin-layer chromatographic technique has been developed and validated for the quantitative measurement of atomoxetine hydrochloride in pharmaceutical formulations and as the bulk drug. It is straightforward, accurate, precise, fast, selective, and repeatable. Performing high performance thin layer chromatography (HPTLC) containing methanol-triethylamine 10:0.5 (v/v) as the mobile phase on aluminium-backed silica gel 60F254 plates, densitometry measurements at 270 mm were performed out. For atomoxetine hydrochloride, this technique produced compact bands (RF 0.55 ± 0.02). The linear calibration plots had a correlation value of 0.9986 and ranged from 300 to 1800 ng per spot. The percentage of recovery fell between 98.10 and 99.85%. The technique can be applied to the analysis of marketed formulations and was validated in compliance with ICH recommendations.[26]

Choi CI et al

The purpose of this work was to validate a sensitive and trustworthy analytical technique using liquid chromatography electrospray ionization tandem mass spectrometry for the pharmacokinetic analysis of atomoxetine in human plasma. A metoprolol internal standard was employed. Using methyl t-butyl ether for liquid-liquid extraction, the supernatant was evaporated. After reconstituting the residue, an aliquot was added to the high-performance liquid chromatographic apparatus. A Phenomenex Luna C18 column (2.0 mm × 100 mm, 3 ?m particles) was used for the separation, and the mobile phase consisted of 10 mM ammonium formate buffer with methanol = 10:90 (v/v). Quantification was done using tandem mass spectrometry in the multiple reaction monitoring mode with electrospray ionization positive ionization. For atomoxetine, the mass transition pairs m/z 256 ? 44 and m/z 268 ? 116 were utilized, respectively. The internal standard and atomoxetine were found to have retention periods of 0.9 and 1.0 minutes, respectively, and the mobile phase flow rate was 0.25 mL/min. With a lower limit of quantification of 1 ng/ml, the atomoxetine calibration curve was linear in the concentration range of 1-750 ng/mL (r2 = 0.9992). For atomoxetine, the accuracy ranged from 93% to 102%. For atomoxetine, the intra- and interday validation yielded coefficients of variation (precision) of 4.0–6.8 and 1.1–9.6%, respectively. After a single oral dose of 40 mg of atomoxetine was given to twelve healthy male volunteers, the pharmacokinetic properties of the medication were assessed. For atomoxetine, the average AUC0–24 h, Cmax, Tmax, and T1/2 were, respectively, 1.9 ± 0.8 ?g h/mL, 0.34 ± 0.11 ?g/mL, 1.0 ± 0.5 h, and 3.9 ± 1.3 h.[27]

Shang DW et al

Using a single 40 mg dose as the starting point, a randomized, open-label, two-period crossover study involving 22 healthy male Chinese subjects was carried out, with a one-week wash-out period, to assess the bioequivalence of a newly developed formulation of atomoxetine hydrochloride (CAS 82248-59-7) capsules (test) and an existing branded capsule. In compliance with Chinese regulatory regulations, this study was created specifically for Honglin Pharmaceutical Co. Ltd. and was contracted to be completed by Beijing Anding Hospital. It was carried out in accordance with SFDA guidelines. Pre- and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 16, and 24 hour post-drug administration was observed for blood sample collection. UV-detected high-performance liquid chromatography (HPLC) was used to measure the quantities of plasma. To determine the pharmacokinetic characteristics and assess the bioequivalence of the two formulations, a noncompartmental technique was employed. The ratios (test/reference) of atomoxetine for AUC0-24, AUC0-?, and Cmax had a 90% confidence interval (CI) of 100.9 % (93.6–108.8 %), 103.1 % (95.1–111.7 %), and 105.2 % (92.8–119.4 %), respectively. These values fell within 80–125 % and 75–133 %. Laboratory results and vital signs showed no abnormalities or alterations that were clinically noteworthy. Based on these findings, it can be said that the atomoxetine capsule test formulation satisfies the regulatory requirement for bioequivalence to the reference formulation.[28]

Stegmann B et al

For the purpose of quantifying methylphenidate (MPH), dexamphetamine (DXA), and atomoxetine in serum and oral fluid, a high-performance liquid chromatography method has been created and verified. Liquid-liquid extraction, derivatization using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride as a label, and chromatographic separation on a Phenomenex Gemini-NX C18 analytical column utilizing gradient elution with water–acetonitrile are the steps in the analytical process. At the excitation wavelength of 330 nm and the emission wavelength of 440 nm, the derivatized analytes were found. Oral fluid samples were taken concurrently with blood samples from patients diagnosed with hyperkinetic condition in order to analyse the oral fluid/serum ratios. Under the same or comparable conditions, the approach permits the measurement of all analytes in serum and oral fluid in less than 16 minutes. The oral fluid/serum ratios for DXA and MPH were quite inconsistent and indicated that these medications were building up in the mouth fluid. Following the administration of therapeutic dosages, the developed approach involves the measurement of MPH, DXA, and atomoxetine concentrations in serum and oral fluid. Samples of oral fluid are helpful for the qualitative detection of DXA and MPH.[29]

Rahman Z et al

Utilizing RP-HPLC, the chromatographic development was completed. The mobile phase in the Xterra RP 18 column (250 mm × 4.6 mm, 5 µ particle size) was made up of 80:20 V/V methanol and water. The effluents were seen at a wavelength of 270 nm, and the flow rate was 1.0 mL/min. It was discovered that the retention time was 5.350 minutes. Regarding linearity, accuracy, precision, and robustness, the procedure was verified in accordance with the International Conference on Harmonization Guidelines. Regression coefficient of 0.9999 indicated that the calibration curve was linear over the range of 2–10 ?g/ml. The technique has demonstrated excellent specificity and sensitivity.[30]

Ravisankar P et al

Chromatographic separation was carried out using a photodiode array detector and a Shimadzu-2010 with a quaternary pump and a Symmetry-C8 column (4.6 mm I’d. X 150 mm, 5 µm particle sizes). A 40:60 v/v mixture of buffer and methanol was employed as the mobile phase, with a flow rate of 1.0 ml/min and a detection wavelength of 271 nm. 3.20 minutes was determined to be the ATX retention time. Between 15 and 105 µg/ml, the calibration was linear (r2 = 1). It was discovered that the limits of detection and quantitation were, respectively, 0.595 ?g/ml and 1.805 µg/ml. The range of 99.25 to 100.91% was found for ATX recovery in tablet formulation. At 96.69% w/w, the percentage test for ATX was discovered.[31]

Faridi N et al

Sensitive analytical techniques are needed to track the drugs trough level. Thus, the trend solvent bar microextraction method in conjunction with HPLC-UV was used to identify traces of this medication. Preconcentration and microextraction of the target analyte were made possible by the use of a pH gradient of 3.0 and 10.3 for the donor and acceptor phases, respectively. The Artificial Neural Network (ANN) correctly modelled the results. The best outcomes were attained after 26 minutes at 25°C with a stirring velocity of 365 rpm and a salt addition of 15.1%. Chromatographic analysis was performed by Shimadzu HPLC system equipped with C18 analytical column (150 mm × 4.6mm, 5?m), a mixture of Phosphate buffer: acetonitrile (70:30, v/v) was used as mobile phase at a 1 ml/min flow rate. The quantification limit was 20 ng mL-1, while the detection limit was 7.0 ng mL-1. With a preconcentration factor of 112 and a coefficient of estimate greater than 0.9972, it provided a good linearity range of 20-5000 ng mL-1. The analysis relative standard deviations were 4.6% for each day (n = 3), and 6.2% for each day (n = 9).[32]

Nataraj KS et al

A 5µm particle-packed Phenomenex 250mm x 4.6mm column effectively created the chromatographic conditions. Pumped at a flow rate of 1.0 millilitre per minute, the ideal mobile phase was trimethylamine (pH -3): acetonitrile (50:50 v/v). AY220 UV detector, Shimadzu LC 2010A HT Auto Sampler, and lab solutions software employ a detection wavelength of 271 nm, 20?l is the injection volume. Regarding specificity, precision, linearity, accuracy, and robustness, the developed technique was validated in accordance with the principles set forth by the International Conference on Harmonization (ICH). For atomoxetine in the concentration range of 10% to 130%, a linear connection between peak areas and concentrations was found. For atomoxetine, the correlation coefficient (R2) is 0.99. % RSD of the intermediate precision was determined to be 0.31 and 0.14, respectively, while % RSD of the accuracy was less than 2. It was discovered that the procedure exhibited resilience, withstood minor intentional modifications in the mobile phase's composition, temperature, and flow rate.[33]

Teichert J et al

For application in neuroscientific research, an HPLC technique from routine TDM measurement of atomoxetine or citalopram in plasma was modified and verified. This is achieved by the use of light diode array detection with UV absorption at 205 nm.  Chromatographic separation was performed on a Zorbax Eclipse XDB C8, 3.5-Micron Narrow-Bore column (2.1 x 150 mm) maintained at 25oC, a mixture of acetonitrile and aqueous 30 mM potassium dihydrogen phosphate (34:66 (v/v), pH 5.1) were used as the mobile phase, at a flow rate of 0.225 mL/min. For both atomoxetine and escitalopram, the approach reported here was found to be linear in the range of 0.002 - 1.4 mg/L and 0.0012 - 0.197 mg/L, with an overall mean intra-day and inter-day imprecision and accuracy bias < 10>

Wassel AA et al

In the current study, atomoxetine and fluoxetine in their prescription dose forms, Strattera® and Prozac®, were determined using a high-performance liquid chromatographic (HPLC) method. Thermo Hypersil BDS C18 column (250 mm x 4.6 mm I.D., 5 µm particle size) was used for the analysis. The mobile phase consisted of 625 ml of Acetonitrile (375:625, v/v) and 0.1 ml of Tetra-n-Butylammonium hydroxide + 0.4 ml triethylamine (pH adjusted to 3.5 with phosphoric acid). The flow rate of the mixture was 1 ml/min, and the UV detection wavelength was 220 nm. The new method demonstrated linearity over the concentration range of 1-16µg/ml (r2 = 0.99999), with atomoxetine and fluoxetine having respective detection limits of 0.028 and 0.065µg/ml and quantitation limits of 0.085 and 0.198µg/ml.[35]

Xia Y

In this work, atomoxetine-d3 was used as the internal standard and an LC-MS/MS method was developed and validated for the measurement of atomoxetine levels in human plasma. Simple protein precipitation using MeOH was used to prepare the samples. With a gradient elution and a flow rate of 0.25 mL min?1, the analyte was separated using a Kinetex C18 column (2.1 mm × 50 mm, 2.6 ?m, Phenomenex). Using a MeOH and water solution with 0.1 mM formic acid and 5 mM ammonium acetate (pH 6.26) as the mobile phase, the issue of uneven retention times between the atomoxetine solution samples and plasma samples was effectively resolved. Using the 256.4 ? 43.8 and 259.3 ? 47.0 transitions for atomoxetine and atomoxetine-d3, respectively, detection was carried out under positive-electrospray-ion multiple reaction-monitoring mode. The range used to obtain linearity in plasma was incredibly broad, ranging from 0.500 to 2000 ng mL?1. The removal of carryover contamination was accomplished with a complicated needle wash solution that contained ACN: MeOH: isopropanol: H2O (4:4:1:1, v/v/v/v).[36]

Wassel AA et al

The development of an atomoxetine rapid resolution liquid chromatographic (RRLC) technology for the medicinal dosage form Strattera ® was reported in the current work. The analysis was achieved on Agilent Eclipse XDB C18 column (50 mm x 4.6 mm I’d, 1.8 ?m particle size) using mixture of aqueous 0.04 M Glacial acetic acid and 0.03M triethylamine- acetonitrile as a mobile phase (42:58, v/v) pH 4.6 at 0.27 mL/min flow rate with UV detection at 220 nm. The developed method was linear over the concentration range of 4-40 ?g /ml (r2 = 0.99969) with a limit of detection and quantitation 0.44 ?g /ml and 1.32 ?g /ml for atomoxetine. The new RRLC technique was validated in terms of quantitation limit, detection limit, specificity, linearity, accuracy, and precision. The statistical analysis proved that the developed method for quantification of atomoxetine as bulk drug and from pharmaceutical preparation is reproducible and selective.[37]

Ceylan B et al

The purpose of this work was to develop a sensitive, quick, and simple ultra-high performance liquid chromatographic method (UHPLC) for measuring the amount of atomoxetine found in different medicinal plants. Salvia officinalis L., Rosmarinus officinalis L., Melissa officinalis L., and Ginkgo biloba L. A reversed phase C18 (5 ?m × 4.6 mm × 150 mm) analytical column, coupled with a mobile phase made up of acetonitrile (50:50 v/v) and monobasic potassium dihydrogen orthophosphate (pH = 6.8) at a flow rate of 0.8 ml/min, and a diode array detector (DAD) that detected at 215 ± 2 nm, were used to achieve the chromatographic separation. The envisioned method's linear behaviour was tested in the 0.5-20 ?g/ml range (r2=0.09990). LOD and LOQ values were determined as 0.16 and 0.5 ?g/ml. It is discovered that the RSD values for both assays' hourly and daily readings are less than 2.5%.[38]

ACKNOWLEDGEMENTS

We express our sincere thanks to the Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College (MMC), Chennai for providing necessary facilities for the research work.

CONFLICTS OF INTEREST

The author declares there is no conflicts of interest.

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