Isocratic Liquid Chromatography Research Articles

A New Method for Quantifying Aluminum in Antiperspirant Lotion Using a Differential Refractometer and an HPLC Column Containing a Maleate Functional Group Kumarswamy Ummiti1*, Narayana Reddy Godi2 , Hanimi Reddy Bapatu2

Aluminum chlorohydrate is a chemical substance employed as an agent to reduce perspiration. As an active antiperspirant agent, it is present in over-the-counter hygiene products at a concentration of up to 25%. The concentration of aluminum chrohydrate will be estimated indirectly by measuring the concentration of free aluminum ions. Various methodological approaches, including atomic absorption spectroscopy, ion chromatography, and titration, can be employed for the estimation of aluminum. In this study, a novel reverse-phase isocratic liquid chromatography approach was created. The method utilized a differential refractometer, a column packed with a maleate group, and an eluent consisting of 2.0% v/v formic acid. The suggested methodology demonstrates a high degree of precision in quantifying the presence of aluminum in Aldry lotion. According to studies testing accuracy, linearity, and precision, the disclosed analytical technique for quantifying aluminum has a range of 0.125 mg/mL to 0.375 mg/mL. The proposed methodology has a significantly reduced execution time of 10 minutes and is characterized by its cost-effectiveness.

Validation of a quantitative liquid chromatography tandem mass spectrometry assay for linezolid in cerebrospinal fluid and its application to patients with HIV-associated TB-meningitis

Tuberculous meningitis treatment outcomes are poor and alternative regimens are under investigation. Reliable methods to measure drug concentrations in cerebrospinal fluid are required to evaluate distribution into the cerebrospinal fluid. A simple and quick method was developed and validated to analyse linezolid in human cerebrospinal fluid. Samples were prepared by protein precipitation followed by isocratic liquid chromatography and tandem mass spectrometry. The run time was 3.5 min. Accuracy and precision were assessed in three independent validation batches with a calibration range of 0.100–20.0 μg/mL. The method was used to analyse cerebrospinal fluid samples from patients with tuberculous meningitis enrolled in a clinical trial. Potentially infective patient samples could be decontaminated using Nanosep® nylon and Costar® nylon filter tubes under biosafety level 3 conditions before analysis. The filtration process did not significantly affect the quantification of linezolid. Linezolid concentration in cerebrospinal fluid obtained from tuberculous meningitis patients ranged from 0.197 μg/mL to 15.0 μg/mL. The ratio between average CSF and plasma linezolid concentrations varied with time, reaching a maximum of 0.9 at 6 h after dosing.

Novel Ultra-Fast Liquid Chromatography Method for Simultaneous Quantification of Metformin Hydrochloride and Remogliflozin Etabonate

An advanced formulation having remogliflozin etabonate (REM) and metformin hydrochloride (MET) for the treatment of diabetes was recently approved. However, there hasn't been any information reported on a UFLC technique for simultaneously quantifying both active substances. Therefore, aim of the study was the development of an ultra-fast isocratic liquid chromatography method for quantification of MET and REM in API and dosage form. The reversed phase-UFLC method was developed with a very short run time of only 3 min with no interference due to blank. The compounds were separated using Shimadzu Sim-pack XR ODS (75 mm × 3 mm, 2.2 μ) column with mobile phase consisting of buffer (0.1% phosphoric acid):acetonitrile (30:70). The developed method was validated as per ICH-Q2(R1). The linearity was established in the range of 0.01-125 ppm and 0.01-25 ppm for MET and REM, respectively. The average assay was found 99.4% for MET and 99.1% for REM. The accuracy was observed 98.29-99.49% for MET and 98.39-99.73% for REM. The proposed method is very fast and simple however precise and accurate. This method is very useful for regular quality control laboratory to quantify MET and REM in API and dosages forms.

Effects of digital processing on repeatability assessment of a multiple reaction monitoring liquid chromatography-tandem mass spectrometry system by ISO 11843-7.

ISO 11843 part 7 (ISO 11843-7) can provide a standard deviation (SD) of area measurements of a target peak through the stochastic behaviors of instrumental noises. The purpose of this study is to demonstrate that ISO 11843-7 can be applied to assess repeatability in an isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS) system without repetitive measurements. The relative standard deviation (RSD) of the peak area of ergosterol picolinyl ester, which was used as an example, on a multiple reaction monitoring (MRM) chromatogram was determined by ISO 11843-7. The RSD by ISO 11843-7 (N = 1) was within a 95% confidence band of the RSD by repetitive measurements (N = 6). Moreover, the effects of digital smoothing, such as moving average, were also examined on the repeatability assessment in LC-MS/MS by ISO 11843-7. From the results of the comparisons of the RSDs obtained by ISO 11843-7 and the repetitive measurements, it was shown that suitable RSDs of the peak area were obtained from the smoothed MRM chromatograms by the moving average for narrow data point windows (e.g., one-sixth of the peak width). In conclusion, the utility of repeatability assessment based on ISO 11843-7 has been expanded for the validation of an LC-MS/MS system.

Stability Indicating RP-HPLC Method Development and Validation for the Determination of Solifenacin Succinate in Bulk and its Pharmaceutical Formulation

The current work aims to establish a novel and advanced reverse phase isocratic liquid chromatography system followed by validation and to conduct stability analysis in active pharmaceutical ingredients and formulations for the quantification of Solifenacin Succinate. The optimized elution was achieved with column Sunfire C8 (4.6 x 150mm, 5µm), using the mobile phase of Buffer: Methanol: Acetonitrile in the composition ratio of 45:45:10 v/v. The wavelength of detection was selected as 220nm with 1.0ml/ min flow rate and 30μl injection volume. The retention time of Solifenacin Succinate was found 2.94 min respectively. The method developed has been validated for various analytical parameters according to ICH guidelines. The Linearity was attained at 20 to100 μg/ml of concentration range. The established method was proved as reproducible. The Assay was obtained as 100.40%. The degradation studies were carried out at all degradative conditions and the results of degradation studies denote that the current method was specific, reliable, and economical. Hence, the developed method can be applied for the qualitative and quantitative determination of the selected drug and its commercial formulations.

DEVELOPMENT AND VALIDATION OF REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ASSAY METHOD FOR ESTIMATION OF FENOFIBRATE IN TABLET DOSAGE FORM PREPARED USING CRYSTALLO-CO-AGGLOMERATES OF THE DRUG

The aim of the present study was to develop a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method and validate for the determination of fenofibrate in tablet dosage forms. RP-HPLC method was developed using Hi Q Sil C18 (250 cm × 4.6 mm, 5 μm) and mobile phase comprising 1 mM ammonium acetate buffer: Acetonitrile (10:90 v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 290 nm. The retention time was found to be 6.15 ± 0.03 min. Validation of the method was performed for precision, accuracy, linearity, robustness, specificity and sensitivity to conform to the International Conference on Harmonization (ICH) guidelines. The data of linear regression analysis indicated a good linear response in the concentration range of 5 μg/mL–30 μg/mL with correlation co-efficient (R2) of 0.997. The developed method was found to be simple, sensitive, accurate and repeatable for assay of tablets of fenofibrate prepared using crystallo-co-agglomerates of the drug.

Simultaneous determination of itraconazole and its CYP3A4-mediated metabolites including N-desalkyl itraconazole in human plasma using liquid chromatography-tandem mass spectrometry and its clinical application

BackgroundItraconazole (ITZ), a triazole antifungal agent, is metabolized to hydroxy-ITZ (OH-ITZ), keto-ITZ (KT-ITZ), and N-desalkyl ITZ (ND-ITZ) by cytochrome P450 3A4. The pharmacokinetics of ND-ITZ remain largely unknown due to the lack of an accurate and reliable determination method. This study aimed to develop a simultaneous determination method for ITZ and its three major metabolites including ND-ITZ in human plasma using isocratic liquid chromatography coupled to tandem mass spectrometry and then apply the method in a clinical setting.MethodsPlasma specimens were pretreated by protein precipitation with acetonitrile. The supernatant was separated on a 3-μm particle octadecyl silane column (75 × 2.0 mm I.D.) in an isocratic elution of acetonitrile and 5 mM ammonium acetate (pH 6.0) (57:43, v/v). The method was applied to 10 patients treated with oral ITZ.ResultsThe calibration curves of ITZ, OH-ITZ, KT-ITZ, and ND-ITZ were linear over the concentration ranges of 15–1500, 15–1500, 1–100, and 1–100 ng/mL, respectively. The pretreatment recoveries and matrix factors were 90.1–102.2% and 99.1–102.7%. Their intra- and inter-assay accuracies and imprecisions were 94.1–106.7% and 0.3–4.4%. The plasma concentrations of ITZ, OH-ITZ, KT-ITZ, and ND-ITZ 12 h after dosing ranged from 32.5–1127.1, 19.0–1166.7, 1.1–5.4, and 3.5–28.3 ng/mL, respectively, in immunocompromised patients.ConclusionsThis study developed a simultaneous determination method for concentrations of ITZ and its three metabolites including ND-ITZ in a clinical setting.

Development and validation of liquid chromatographic methods for the estimation of the acceptance values of some hazardous preservatives in pharmaceutical formulations. A comparative study

Gradient and isocratic liquid chromatography were applied for the simultaneous determination of three preservatives, namely; benzyl alcohol (BA), methyl paraben (MP) and propyl paraben (PP). Separation for both elution modes was carried out using a C18 column kept at 60°C throughout the analysis, accompanied with a flow rate of 2 mL/min and UV detection at 254 nm. Gradient liquid chromatography used a time programme with two solvents (0.1% formic acid and acetonitril), while isocratic elution was performed with 0.1% formic acid: 2-propanol in a ratio of 70:30, v/v. Full validation study was conducted for both separation modes, where both of them were linear over the concentration ranges of 100.0–2000.0, 2.5–100.0, and 2.0–200.0 µg/mL for BA, MP and PP respectively. Using gradient elution, the detection limits for BA, MP, and PP were found to be 50.0, 1.8, and 1.5 µg/mL respectively. The corresponding values in isocratic elution were 60.0, 1.5, and 1.3 µg/mL. On the other hand, the limit of quantification for BA, MP, and PP using gradient mode are 70.0, 2.0, and 1.8 µg/mL respectively. While isocratic elution resulted in corresponding values of: 80.0, 2.3, and 1.7 µg/mL. The accuracy of the proposed method was illustrated from the high percentage recoveries values which were 99.67%, 100.42%, and 99.85% for BA, MP, and PP respectively using gradient mode, with corresponding values of 99.79%, 100.39%, and 99.87% for the isocratic elution mode. In addition, gradient elution was further applied to detect and assay the selected preservatives in different pharmaceutical formulations demonstrating that their quantities are within the stated ranges, and eventually, a comparative overview was introduced.

Validated LC-MS/MS Method for the Simultaneous Determination of Amlodipine and Its Major Metabolites in Human Plasma of Hypertensive Patients

No information on the pharmacokinetic characteristics of amlodipine (AML) metabolites is available. This study aimed to develop a method based on isocratic liquid chromatography coupled to tandem mass spectrometry for the simultaneous determination of AML and its 2 major metabolites, dehydroamlodipine (DH-AML) and O-des[2-aminoethyl]-O-carboxymethyl DH-AML (CM-DH-AML), and to use it for monitoring this drug in hypertensive patients. Acetonitrile-deproteinized plasma specimens were separated using an octadecyl-silica column (3-μm particle size) with a mobile phase consisting of 50% methanol containing 0.15% of formic acid in water. The run time was 9 minutes. The mass spectrometer was run in the positive ion electrospray ionization mode. This method was applied for the determination of AML and its metabolites in plasma samples from patients treated with this drug. The calibration curves in human plasma of AML, DH-AML, and CM-DH-AML were linear over the concentration ranges of 0.5-64, 1-64, and 0.5-64 ng/mL, respectively, and their lower limits of quantification were 0.5, 1, and 0.5 ng/mL, respectively. Their extraction recovery rates and matrix factors in human plasma were 94.8%-109.0% and 97.0%-101.4%, respectively. The intra-assay and interassay imprecisions and accuracies were within 10.8% and 95.4%-111.2%, respectively. The plasma concentration ranges of AML, DH-AML, and CM-DH-AML were 6.5-20.9, 1.4-10.9, and 5.6-38.3 ng/mL, respectively. The present method with acceptable analytical performance can be helpful for monitoring the plasma concentration of AML, including the determination of its metabolites in patients with hypertension.

Validated LC–MS/MS Method for Simultaneous Determination of Aripiprazole and its Three Metabolites in Human Plasma

Aripiprazole, a partial agonist of dopamine D2 receptors, is primarily metabolized to active dehydroaripiprazole and then converted to its urinary metabolites 1-(2,3-dichlorophenyl) piperazine (DCPP) and 3,4-dihydro-7-(3′carboxy) propoxy-2(1H) quinolinone (DCPQ). This study aimed to develop a simultaneous determination method for aripiprazole and its three metabolites in human plasma using isocratic liquid chromatography coupled to tandem mass spectrometry and then to apply it to pharmacokinetic analyses. The pretreatments for human plasma involve protein precipitation using acetonitrile under basic conditions. Aripiprazole and its metabolites were separated on an octadecylsilyl column filled with 3-µm particles using an isocratic mixture of 30% acetonitrile containing 0.1% formic acid with a total run time of 10 min. The mass spectrometer was run in the positive ion multiple reaction monitoring mode. The calibration curves were linear over the plasma concentration ranges 2–2000 ng mL−1 for aripiprazole, and 1–1000 ng mL−1 for dehydroaripiprazole, DCPP, and DCPQ. The lower limits of quantification in human plasma were 2 ng mL−1 for aripiprazole and 1 ng mL−1 for the metabolites. Their pretreatment recoveries were 88.3–102.7%. The intra-assay precision and accuracy were within 1.7–9.6 and 86.9–105.9% and inter-assay precision and accuracy were within 3.1–9.1 and 87.4–102.4%, respectively. The plasma concentrations of aripiprazole, dehydroaripiprazole, DCPP, and DCPQ ranged from 42.8 to 1369, 1.01–548, 1.02–46.1, and 1.69–475 ng mL−1, respectively, in 20 schizophrenia patients. In conclusion, this simultaneous determination method with acceptable analytical performance can be helpful for evaluating the pharmacokinetics of aripiprazole including the determination of its metabolites, in clinical research.

A Liquid Chromatography Method for Simultaneous Determination of Diphenhydramine, Promethazine, Chlorpheniramine and Ephedrine in Cold-Cough Syrups

Asimple, rapid isocratic liquid chromatography method was developed for the simultaneous determination of diphenhydramine, promethazine, chlorpheniramine, and ephedrine in cold-cough syrups commonly available in the Kenyan market. The influence of the percentage of organic modifier, ion pairing agent, buffer concentration as well as pH and column temperature on the selectivity with respect to analytes was investigated. Optimum chromatographic separation was achieved using a C18 Gemini® NX column (250 mm × 4.6 mm, 5 μm) maintained at 40°C and a mobile phase comprising methanol – triethylamine – 0.2 M ammonium acetate pH 5.0 – water mixture (50 : 0.15 : 40 : 9.85, v/v) delivered at a flow rate of 1.0 mL/min. Upon validation, the proposed liquid chromatography method satisfied the International Committee on Harmonization acceptance criteria for linearity, sensitivity, precision, and robustness. The method was applied in the analysis of commercial samples obtained from Nairobi County, Kenya. The method can be used in routine analysis of cold-cough syrups containing the specified compounds.

Validated determination method of tramadol and its desmethylates in human plasma using an isocratic LC-MS/MS and its clinical application to patients with cancer pain or non-cancer pain.

BackgroundThis study aimed to develop a simultaneous determination method for tramadol and its desmethylates in human plasma using isocratic liquid chromatography coupled to tandem mass spectrometry and to validate it for pharmacokinetic evaluation in patients with cancer pain or non-cancer pain.MethodsThe pretreatments for human plasma involved protein precipitation using acetonitrile and methanol under basic conditions. Tramadol, O-desmethylate, N-desmethylate, and N,O-didesmethylate were separated on an octadecylsilyl column filled with 3-μm particles using isocratic mixture of methanol and 0.15 % formic acid in water (35:65, v/v). The mass spectrometer was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of plasma samples in patients treated with oral tramadol.ResultsThe chromatographic total run time was 10 min. The calibration curves in human plasma of tramadol, O-desmethylate, N-desmethylate, and N,O-didesmethylate were linear over the concentration ranges of 12.5–1600, 2.5–320, 2.5–320, and 2.5–320 ng/mL, respectively. The lower limits of quantitation of tramadol and its desmethylates in human plasma were 12.5 and 2.5 ng/mL. Their extraction recoveries were 85.5–106.3 %. The intra-day and inter-day precisions and accuracies were 1.6–10.2 % and 89.2–106.2 % for all analytes. The plasma concentration ranges of tramadol, O-desmethylate, N-desmethylate, and N,O-didesmethylate were 18.2–564, 11.8–137, 4.9–250, and 6.1–147 ng/mL in cancer patients, and 32.8–670, 7.0–84.8, 5.1–317, and 6.7–85.2 ng/mL, respectively, in non-cancer patients.ConclusionsThe present method with acceptable analytical performance can be helpful for evaluating the pharmacokinetics of oral tramadol, including the determination of its desmethylates, for patients with cancer pain or non-cancer pain in clinical settings.

Simultaneous analysis of heparosan oligosaccharides by isocratic liquid chromatography with charged aerosol detection/mass spectrometry

Uncovering the biological roles of heparosan oligosaccharides requires a simple and robust method for their separation and identification. We reported on systematic investigations of the retention behaviors of synthetic heparosan oligosaccharides on porous graphitic carbon (PGC) column by HPLC with charged aerosol detection. Oligosaccharides were strongly retained by PGC material in water-acetonitrile mobile phase, and eluted by trifluoroacetic acid occurring as narrow peaks. Addition of small fraction of methanol led to better selectivity of PGC to oligosaccharides than acetonitrile modifier alone, presumably, resulting from displacement of methanol to give different chemical environment at the PGC surface. Van’t-Hoff plots demonstrated that retention behaviors highly depended on the column temperature and oligosaccharide moieties. By implementing the optimal MeOH content and temperature, a novel isocratic elution method was successfully developed for baseline resolution and identification of seven heparosan oligosaccharides using PGC-HPLC–CAD/MS. This approach allows for rapid analysis of heparosan oligosaccharides from various sources.

Simultaneous separation and determination of four main isoflavonoids in Astragali Radix by an isocratic LC/ESI-MS method

A simple, reliable and rapid isocratic liquid chromatography (LC)-mass spectrometric detection (MS) coupled with electrospray ionization (ESI) method for simultaneous separation and determination of calycosin-7-O-β-D-glucoside, ononin, calycosin and formonometin in Astragali Radix was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using water and acetonitrile (70:30, v/v) containing 0.2% (v/v) acetic acid as a mobile phase and a 2.0 mm×150 mm Hypersil-Keystone C18 column. Selective ion monitoring (SIM) mode and [M+H]+ ions at m/z 447, 431, 285 and 269 were used for quantitative analysis of four main active components above mentioned. The calibration curves were linear in the range of 0.4−175.0 μg/mL for calycosin-7-O-β-D-glucoside, 0.2−146.0 μg/mL for ononin, 0.4−210.0 μg/mL for calycosin and 0.5−217.0 μg/mL for formonetion, respectively. The limits of quantification (LOQ) and detection (LOD) were 0.4 μg/mL and 0.08 μg/mL for calycosin-7-O-β-D-glucoside, 0.2 μg/mL and 0.06 μg/mL for ononin, 0.4 μg/mL and 0.1 μg/mL for calycosin, 0.5 μg/mL and 0.1 μg/mL formonetion, respectively. The standard recoveries were in the range of 96.5%−104.7%. The developed method has successfully been used for the determination of four main flavonoids in Astragali Radix from various sources and can be used for identification, differentiation and quality evaluation of Astragali Radix.

Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma, including metabolite concentrations at steady state.

A novel, rapid and sensitive liquid chromatography tandem-mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi-quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C18 column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi-quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5-100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.

Separation Speed and Power in Isocratic Liquid Chromatography: Loss in Performance of Poppe vs Knox-Saleem Optimization.

The best separation possible at a given analysis time and maximum system pressure is achieved by simultaneously optimizing column length, eluent velocity, and particle size. However, this three-parameter optimization is rarely practicable because only a few commercially available particle sizes exist. Practical optimization for systems described by the van Deemter equation therefore proceeds by first selecting an available particle size and then optimizing eluent velocity and column length. This two parameter ("Poppe") optimization must result in poorer performance with respect to both speed and efficiency because one fewer degree of freedom is used. A deeper analysis identifies a distinct point on each pair of "Poppe" curves beyond which the more efficient (and faster) separation is maintained by changing from smaller to larger particles. Here, we present simple equations identifying these "crossover points" in terms of analysis time and plate count thereby allowing a practitioner to rapidly identify the correct particle size for use in tackling a particular separation problem. Additionally, we can now quantitatively compare two-parameter and three-parameter optimization. Surprisingly, we find that for systems well-described by the van Deemter equation there is little separating power lost (only about 11% in the worst case) as a result of the limited availability of different particle sizes in using two-parameter optimization when compared to the ideal three-parameter optimization so long as one changes particle size at the prescribed crossover points. If these crossover times are not used, a great deal of separating power will be needlessly lost.

Stability-indicating liquid chromatographic and UV spectrophotometric methods for the quantification of ciprofibrate in capsules and tablets

This study describes the development and evaluation of stability-indicating liquid chromatographic (LC) and UV spectrophotometric methods for the quantification of ciprofibrate (CPF) in tablets and capsules. Isocratic LC separation was achieved on a RP18 column using a mobile phase of o-phosphoric acid (0.1% v/v), adjusted to pH 3.0 with triethylamine (10% v/v) and acetonitrile (35:65 v/v), with a flow rate of 1.0 mL min-1. Detection was achieved with a photodiode array detector at 233 nm. For the spectrophotometric analysis, ethanol and water were used as the solvent and a wavelength of 233 nm was selected for the detection. The methods were validated according to International Conference on Harmonization (ICH) guidelines for validating analytical procedures. Statistical analysis showed no significant difference between the results obtained by the two methods. The proposed methods were successfully applied to the CPF quality-control analysis of tablets and capsules.

Development and validation of three methods: liquid chromatography, flow injection analysis and UV spectrophotometry for the routine control of nystatin capsules

ObjectivesAs an alternative to amphotericin B used for selective digestive decontamination, physicians asked the hospital pharmacy for the preparation of nystatin capsules 500 000 IU.MethodsThree methods were considered for the...

Further Considerations of Exact Equations for Peak Capacity in Isocratic Liquid Chromatography

Further consideration is made of the exact equation for peak capacity by Potts, L. W.; Carr, P. W. Anal. Chem. 2011, 83, 7614-7615 for liquid chromatographs with significant extra-column broadening. Equivalent exact equations are easily derived that provide comprehension and insight of how extra-column broadening degrades chromatographic performance as measured by peak capacity.

Ultra-High Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for the Determination of Caffeine in Energy Drinks

A simple and highly sensitive method for the determination of caffeine in energy beverages was developed and validated. Sample preparation utilizing solid-phase extraction (SPE) was simple and reliable. Separation by isocratic ultra-high performance liquid chromatography (UPLC) with a reversed-phase C18 column was performed within 6 min. The use of SPE with UPLC coupled with electrospray ionization-multiple tandem mass spectrometry detection (ESI-MSn) was accurate, reproducible, and validated for the determination of caffeine in energy drink matrices. The limit of quantification for caffeine was approximately 2.1 ng mL−1. The intra-day and inter-day precisions were less than 4%, and the accuracy of the measurements was between 85.1% and 93.2%. Results for caffeine concentrations in eighteen beverages were compared to the values on the labels. This paper describes the first use of the UPLC–ESI-ion trap MSn technique for quality-control purposes of caffeine present in energy drinks.