Styrene‐N‐phenylacrylamide co‐polymer modified silica monolith particles with an optimized mixing ratio of monomers as a new stationary phase for the separation of peptides in high performance liquid chromatography

A stationary phase was prepared by chemical derivatization of the support particles with a layer of copolymer composed of styrene and N‐phenyl acrylamide. Silica monolith particles of ca. 2.6 µm (volume‐based average) have been prepared as the support particles by sol‐gel reaction followed by differential sedimentation. The particles were reacted with 3‐chloropropyl trimethoxysilane followed by sodium diethyldithiocarbamate to introduce an initiator moiety. Then, the copolymer layer was immobilized via reversible addition‐fragmentation transfer polymerization. The resultant phase was packed in glass‐lined stainless‐steel micro‐columns (1 x 150 mm) and evaluated for the separation of a mixture composed of five peptides (Trp‐Gly, Thr‐Tyr‐Ser, angiotensin I, isotocin and bradykinin). The effect of monomer mixing ratio (styrene versus N‐phenyl acrylamide) on the chromatographic separation efficiency of the stationary phase was examined. A number of theoretical plates (N) as high as 33 600 plates/column (224 000 plates/m, 4.46 µm plate height) was achieved using the column packed with the optimized stationary phase. The column‐to‐column reproducibility based on three columns packed with three different batches of stationary phase was found satisfactory in separation efficiency, retention factor, and asymmetry factor.     

Determination of levetiracetam in human plasma by online heart‐cutting liquid chromatography: Application to therapeutic drug monitoring

Levetiracetam is an antiepileptic drug for the treatment of psychiatric patients. In this study, a selective, straightforward, and rapid online heart‐cutting liquid chromatography method was developed for the therapeutic drug monitoring of levetiracetam. This method allows for the determination of levetiracetam in human plasma without complex sample preparation. The mobile phases consisted of 30 mM aq. orthophosphoric acid solution/methanol (70:30) at a flow rate of 1 mL/min for the first system and 10 mM aq. orthophosphoric acid solution/methanol (55:45) at a flow rate of 1 mL/min for the second system. The first separation was carried out on a GL Sciences Intersil ODS‐3 column (4.6 mm × 150 mm, 3 µm) and the second separation was carried out on a Restek Ultra PFPP column (4.6 mm × 150 mm, 5 µm). The detection was carried out at 205 nm for both systems. The method was validated for selectivity and linearity, which were in the 6–60 µg/mL range. Intra‐ and interassay accuracies were <112.6%, and the intra‐ and interassay precisions were <6.4% for all quality control samples. The lower limit of quantitation was 6 µg/mL. The developed method was successfully applied for therapeutic drug monitoring of plasma samples from patients.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Ultra high performance liquid chromatography method development for separation of omeprazole and related substances on core‐shell columns using a Quality by Design approach

An updated and improved method for analysis of omeprazole/esomeprazole and related substances on core‐shell columns was developed using Fusion LC Method Development?. The method was optimized with respect to column type, column temperature, mobile phase pH level, and gradient time. Four different core‐shell columns were examined to develop a method suitable for both high performance‐ and ultra‐high performance liquid chromatography using a Quality by Design approach. The final method offers two alternative columns: Poroshell EC C18 (3.0 × 100 mm, 2.7 µm) or Poroshell HPH (3.0 × 100 mm, 2.7 µm) with the same gradient elution condition and mobile phase composition. Total run time is 18 min with 12 min of gradient elution. Phosphate buffer (15 mM, pH 7.8) is selected as the aqueous mobile phase and acetonitrile as the organic mobile phase. Column temperature is set at 40°C and ultraviolet detection at 302 nm. Furthermore, by studying parameters in a systematic way, an understanding of the effect of the input parameters enhances the method robustness and should allow for regulatory flexibility in terms of post‐approval changes. Compared to the current United States Pharmacopeia method, the updated method is faster, more efficient and performs well above acceptance criteria.     

Rapid and sensitive LC‐MS method for pharmacokinetic study of vinorelbine in rats

A rapid and sensitive LC‐electrospray ionization‐MS method was developed for determining vinorelbine in rat plasma. A 100 µL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS‐3 C₁₈ (2.1 × 50 mm, 5 µm) column. The selected ion monitoring ions [M + H]⁺ were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1–1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.     

Rapid high‐performance liquid chromatography–tandem mass spectrometry method for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma and its application in comparative bioavailability study

A rapid high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma using fluoxetine as an internal standard. In the liquid–liquid extraction method, compounds and internal standard were extracted from plasma using methyl tertiary butyl ether as an extraction solvent. The HPLC separation of the analytes was performed on a Zorbax SB‐C₁₈, 50 × 4.6 mm, 5 µm column, using a isocratic elution program using a mobile phase consisting of HPLC‐grade methanol: 5 mm ammonium acetate (80:20 v/v) at a flow‐rate of 1.0 mL/min with a total runtime of 3.0 min. The proposed method has been validated with a linear range of 4–400 ng/mL for venlafaxine and 5–500 ng/mL for O‐desmethyl venlafaxine. The method was applied for a bio‐equivalence study of 75 mg tablets formulation in 32 Indian male healthy subjects under fasting conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of an improved online comprehensive hydrophilic interaction chromatography × reversed‐phase ultra‐high‐pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis

In this study, an improved online comprehensive two‐dimensional liquid chromatography platform coupled to tandem mass spectrometry was developed for the analysis of complex polyphenolic samples. A narrowbore hydrophilic interaction chromatography column (150 × 2.0 mm, 3.0 μm, cross‐linked diol) was employed in the first dimension, while a reversed‐phase column based on monodisperse sub‐2 μm fully porous particles (50 × 3.0 mm, 1.9 μm d.p.) with high surface area (410 m²/g) was employed in the second dimension. The combination of a trapping column modulation interface with the high retentive fully porous monodisperse reversed‐phase column in the second dimension resulted in higher peak capacity values (1146 versus 867), increased sensitivity, sharper and more symmetrical peaks in comparison with a conventional loop‐based method, with the same analysis time (70 min). The system was challenged against a complex polyphenolic extract of a typical Italian apple cultivar, enabling the simultaneous separation of multiple polyphenolic classes, including oligomeric procyanidins, up to degree of polymerization of 10. Hyphenation with an ion trap time‐of‐flight mass spectrometer led to the tentative identification of 121 analytes, showing how this platform could be a powerful analytical tool for the accurate profiling of complex polyphenolic samples.     

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose‐based chiral stationary phase in hydrophilic interaction liquid chromatography mode

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high‐performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 μm particles of cellulose‐based Chiralpak IC‐3 chiral stationary phase. Using a mobile phase containing acetonitrile–water–diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)‐enantiomer impurity of 44.5 and 133.6 ng/mL.     

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4‐O‐deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 µL plasma aliquot was protein precipitated with acetonitrile–methanol (1:1, v/v) containing the internal standard vinorelbine‐d3 and 20 µL volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using isocratic elution with 1 mm ammonium acetate–ammonia buffer pH 10.5–acetonitrile–methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4‐O‐deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 µL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4‐O‐deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre‐)clinical pharmacologic studies with vinorelbine in humans and mice. Copyright © 2009 John Wiley & Sons, Ltd.     

Two high‐performance liquid chromatography–tandem mass spectrometry methods for determination of edaravone and taurine in human plasma: Application to drug–drug interaction and pharmacokinetic studies

Two high‐performance liquid chromatography–tandem mass spectrometry methods were developed and validated for the quantification of edaravone (method A) or taurine (method B) in human plasma. After protein precipitation, separations were achieved on an Ultimate XB‐C8 (2.1 × 50 mm, 3.0 µm) column for edaravone and a ZORBAX SB‐Aq column (2.1 × 100 mm, 3.5 µm) for taurine, respectively. The detection used electrospray ionization source via multiple reaction monitoring in positive‐ion mode for edaravone and negative‐ion mode for taurine, respectively. The lower limits of quantification were 10.0 ng/mL for edaravone and 3.00 μg/mL for taurine. The selectivity, accuracy, and precision of the methods were all within acceptable limits. Two methods were successfully applied to a drug–drug interaction study and a pharmacokinetic study of edaravone and taurine in healthy Chinese volunteers after intravenous infusion of single or compound injection. The results showed that co‐administration of edaravone with taurine increased the C_max and AUC_0‐24 of taurine in human plasma while taurine did not affect the systemic exposure of edaravone. Edaravone and taurine have the dose‐dependent pharmacokinetic profiles in human.     

Development and validation of a stability‐indicating HPLC method for topiramate using a mixed‐mode column and charged aerosol detector

The analysis of topiramate in the presence of its main degradation products is challenging due to the absence of chromophore moieties and their wide range of polarity. Mixed‐mode chromatography has been used in such cases because it combines two or more modes of separation. Charged aerosol detector is also an alternative since its detection is independent of optical properties and analyte ionization. This study is aimed to develop and validate two new stability‐indicating methods by high‐performance liquid chromatography for the main degradation products of topiramate using mixed‐mode chromatography and a charged aerosol detector. Method 1 employed an Acclaim Trinity P1® column (3.0 mm × 150 mm, 2.7 μm) with a mobile phase comprising of 80% ammonium acetate buffer (20 mM, pH 4.0) and 20% methanol at a flow rate of 0.5 mL/min at 35°C. Method 2 utilized a C18 Acclaim 120® column (4.6 mm × 250 mm; 5 μm) with ACN/water (50:50) at a flow rate of 0.6 mL/min at 50°C. Validation of the two methods demonstrated excellent performance with respect to linearity, precision, accuracy, and selectivity. The limits of detection for topiramate, fructose, sulfate, sulfamate, and compound A were 2.97, 12.08, 4.02, 13.91, and 3.94 μg/mL, respectively.     

Fast separation of native proteins using sub‐2 µm nonporous silica particles in a chromatographic cake

A novel method for the fast separation of native proteins was investigated using sub‐2 µm nonporous silica packing inside a chromatographic cake having a diameter much larger than its thickness. Various silica‐based particles ranging from 630 nm to 1.2 µm were synthesized and chemically modified with polyethylene glycol 600. The packing material was laterally packed into a series of chromatographic cakes containing the same diameter (10 mm) and different thicknesses, ranging from 2 to 10 mm, and tested by hydrophobic interaction chromatography. The results showed that the sub‐2 µm NPS particles in a small chromatographic cake were found to have a high efficiency at a flow rate of 10 mL/min and a backpressure of <20 MPa. The effect of the thickness of the chromatographic cake on the resolution of the proteins was also investigated and it was found that too short a column length could dramatically decrease the protein resolution; the minimum column length was also qualitatively evaluated. The presented method is expected to be useful for routine analysis of native and/or intact proteins in hospitals and as a tool for the fast screening protein drugs and optimization of experimental laboratory conditions. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by isocratic high‐pressure liquid chromatography with post‐column hydrolysis and fluorescence detection

A selective, sensitive and rapid high‐performance liquid chromatography method with post‐column hydrolysis and fluorescence detection was developed for the simultaneous quantification of acetylsalicylic acid and its metabolite salicylic acid in human plasma. Following the addition of 2‐hydroxy‐3‐methoxybenzoic acid as internal standard and simple protein precipitation with acetonitrile, the analytes were separated on a ProntoSIL 120 C₁₈ ace‐EPS column (150 × 2 mm, 3 µm) protected by a C₈ guard column (5 µm). The mobile phase, 10 mm formic acid in water (pH 2.9) and acetonitrile (70:30, v/v), was used at a flow rate of 0.35 mL/min. After on‐line post‐column hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) by addition of alkaline solution, the analytes were measured at 290 nm (λ_ex) and 400 nm (λ_em). The method was linear in the concentration ranges between 0.05 and 20 ng/μL for both ASA and SA with a lower limit of quantification of 25 pg/μL for SA and 50 pg/μL for ASA. The limit of detection was 15 pg/μL for SA and 32.5 pg/μL for ASA. The analysis of ASA and SA can be carried out within 8 min; therefore this method is suitable for measuring plasma concentrations of salicylates in clinical routine. Copyright © 2012 John Wiley & Sons, Ltd.     

HPLC quantification of 5‐hydroxyeicosatetraenoic acid in human lung cancer tissues

A simple and cost‐effective HPLC method was established for quantification of 5‐hydroxyeicosatetraenoic acid (5‐HETE) in human lung cancer tissues. 5‐HETE from 27 patients' lung cancer tissues were extracted by solid‐phase extraction and analyzed on a Waters Symmetry C₁₈ column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of methanol, 10 mm ammonium acetate, and 1 m acetic acid (70:30:0.1, v:v:v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 240 nm. The calibration curve was linear within the concentration range from 10 to 1000 ng/mL (r² > 0.999, n = 7), the limit of detection was 1.0 ng/mL and the limit of quantitation was 10.0 ng/mL for a 100 µL injection. The relative error (%) for intra‐day accuracy was from 93.14 to 112.50% and the RSD (%) for intra‐day precision was from 0.21 to 2.60% over the concentration range 10–1000 ng/mL. By applying this method, amounts of 5‐HETE were quantitated in human lung cancer tissues from 27 human subjects. The established HPLC method was validated to be a simple, reliable and cost‐effective procedure that can be applied to conduct translational characterization of 5‐HETE in human lung cancer tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Porphyrinogen fragmentation profiles by ultra‐high‐performance liquid chromatography/electrospray ionisation tandem mass spectrometry

An ultra‐high‐performance liquid chromatography/electrospray ionisation tandem mass spectrometry system is described for the separation and characterisation of uroporphyrinogen, heptacarboxylic acid porphyrinogen, hexacarboxylic acid porphyrinogen, pentacarboxylic acid porphyrinogen and coproporphyrinogen. The separation was carried out on a 100 mm × 2.1 mm Thermo‐Hypersil BDS column (2.4 µm average particle size) by gradient elution with a mixture of acetonitrile, methanol and 1 mol/L aqueous ammonium acetate buffer, pH 5.16, as eluent. The fragmentation pattern of each compound was established by collision‐induced dissociation tandem mass spectrometry. The most characteristic fragmentation was ring opening at one of the four methylene bridges of the protonated porphyrinogen molecule followed by further cleavages of methylene bridges linking the four pyrrole rings at various points to give product ions with methylenepyrrolenine, methylene‐dipyrrolenine and methylene‐tripyrrolenine structures. Copyright © 2011 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of THC, 11‐hydroxy‐THC and 11‐nor‐9‐carboxy‐THC in whole blood by ultra‐performance liquid chromatography/tandem mass spectrometry

A qualitative and quantitative analytical method was developed for the simultaneous determination of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (11‐OH‐THC) and l1‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THC‐COOH) in whole blood. The samples were prepared by solid‐phase extraction followed by ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis using positive ion electrospray ionization and multiple reaction monitoring. The chromatographic separation was performed with an Acquity UPLC® HSS T3 (50 × 2.1 mm i.d., 1.8 µm) reversed‐phase column using a methanol/2 mM ammonium formate (formic acid 0.1%) gradient in a total run time of 9.5 min. MS/MS detection was achieved with two precursor‐product ion transitions per substance. The method was fully validated, including selectivity and capacity of identification, according to the identification criteria (two transitions per substance, signal‐to‐noise ratio, relative retention time and ion ratio) without the presence of interferences, limit of detection (0.2 µg/L for THC and 0.5 µg/L for 11‐OH‐THC and THC‐COOH), limit of quantitation (0.5 µg/L for all cannabinoids), recovery (53–115%), carryover, matrix effect (34‐43%), linearity (0.5‐100 µg/L), intra‐assay precision (CV < 10% for the relative peak area ratios and <0.1% for the relative retention time), inter‐assay accuracy (mean relative error <10%) and precision (CV <11%). The method has already been successfully used in proficiency tests and subsequently applied to authentic samples in routine forensic analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Ultra‐high pressure LC for astaxanthin determination in shrimp by‐products and active food packaging

Nowadays, there is increasing interest in natural antioxidants from food by‐products. Astaxanthin is a potent antioxidant and one of the major carotenoids in crustaceans and salmonids. An ultra‐high pressure liquid chromatographic method was developed and validated for the determination of astaxanthin in shrimp by‐products, and its migration from new packaging materials to food simulants was also studied. The method uses an UPLC® BEH guard‐column (2.1 × 5 mm, 1.7 µm particle size) and an UPLC® BEH analytical column (2.1 × 50 mm, 1.7 µm particle size). Chromatographic separation was achieved using a programmed gradient mobile phase consisting of (A) acetonitrile–methanol (containing 0.05 m ammonium acetate)–dichloromethane (75:20:5, v/v/v) and (B) ultrapure water. This method was evaluated with respect to validation parameters such as linearity, precision, limit of detection, limit of quantification and recovery. Low‐density polyethylene films were prepared with different amounts of the lipid fraction of fermented shrimp waste by extrusion, and migration was evaluated into food simulants (isooctane and ethanol 95%, v/v). Migration was not detected under the tested conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

High‐performance liquid chromatographic column packings with different particle sizes: Chromatographic behavior for the quality analysis of HuanglianShangqing pill

The chromatographic separation of traditional Chinese medicines is still a highly challenging task in analytical science with respect to its hundreds and thousands of chemical compounds, while increase of separation efficiency can greatly improve the separation power of chromatographic column for traditional Chinese medicine. In this study, 13 bioactive components in HuanglianShangqing pill were selected as an index to optimize the separation conditions and evaluate the system suitability of three commercially available columns packed with 1.8, 3.5, and 5.0 μm particles. The chromatographic separations were obtained by the most appropriate Eclipse Plus C₁₈ column (100 × 2.1 mm, 3.5 μm) within 45 min using gradient elution with aqueous‐ammonium acetate (10 mmol/L, pH 5.0) and acetonitrile, at a flow rate of 0.3 mL/min and an operating temperature of 30°C. The quality of HuanglianShangqing pill was assessed through combining simultaneous quantification of 13 compounds with fingerprint analysis. For the qualitative analysis, mass spectrometry was used to confirm the 13 compounds. All the validation data conformed to the acceptable requirements. For the fingerprint analysis, 32 peaks were selected as the common peaks at 254 nm to evaluate the similarities among HuanglianShangqing pills obtained from ten manufacturers.