Single High-performance Liquid Chromatography Research Articles

Experimental design approach for the quantitative analysis of multicomponents by single marker and HPLC fingerprinting of Thunbergia laurifolia aqueous extract.

Thunbergia laurifolia is used in traditional Thai medicine to reduce fever and treat mouth ulcers. However, the quantitative analysis of chemical markers has not yet been officially defined. The objective of this study is to develop a high-performance liquid chromatography (HPLC) method using a design of experiment (DoE) for the quantitative analysis of multicomponents by single marker (QAMS) and fingerprinting of the T. laurifolia aqueous extract. Critical variables were screened using a two-level fractional factorial design, followed by the optimization of the selected variables using a central composite design. The validated method was applied for quality assessment based on QAMS and fingerprinting of the extract. Optimum conditions of DoE for the analysis of caffeic acid, vicenin-2, and rosmarinic acid were determined. The relative correction factors for caffeic acid and vicenin-2 were calculated using rosmarinic acid as an internal reference standard, and their contents in 30 samples were determined. The differences between the external standard method (ESM) and QAMS were compared. No significant difference was observed in the quantitative determination, proving the consistency QAMS and ESM. HPLC fingerprints of T. laurifolia were established with 8 of 12 characteristic peaks that were structurally characterized using HPLC-diode array detection-electrospray ionization/tandem mass spectrometry. The similarity of the fingerprints in all samples was ≥0.74, and the pattern recognition of the characteristic peaks was satisfied. The proposed method efficiently detected multiple components of the T. laurifolia extract. Thus, the method is beneficial in providing references for enhancing the quality control of other herbal medicines.

Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion.

Stereoinversion of Ser residues within proteins, which has been identified in long-lived proteins, influences protein function. To quantify the stereoinversion of Ser residues, we investigated the potential adaptation of our direct peptide analytical method originally established for analyzing the isomerization of asparaginyl/aspartyl residues. Peptide pairs containing L-Ser or D-Ser residues with lengths of four or five residues were synthesized. Separation conditions for these peptide pairs were systematically examined by precisely adjusting the pH of the elution solvent using reverse-phase high-performance liquid chromatography (HPLC). Optimal separation conditions were successfully developed for all peptide pairs, enabling the direct quantification of Ser residue stereoinversion through a single HPLC run. Subsequently, the degree of Ser stereoinversion within the model peptide, Gly-Ser-Gly-Tyr, was determined using the method established in this study. Surprisingly, the stereoinversion of Ser residues occurred only when the absolute configurations of Ser and Tyr residues of the peptide differed from each other, whereas no stereoinversion was observed when their absolute configurations were identical. The experiments using peptides similar to the model peptide reveal that both the N-terminal amino group and the hydroxyl group of the C-terminal Tyr residue are involved in the stereoinversion of the Ser residue. By applying a simple method to quantify the stereoinversion of Ser residues, valuable insights into the mechanisms governing these stereoinversions were obtained.

Simultaneous determination of praziquantel, fipronil, eprinomectin, (S)-methoprene, their key related substances and butylated hydroxytoluene (BHT) in a topical veterinary drug product by a single stability indicating high performance liquid chromatography method

A simple, robust and QC (quality control) friendly high performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of four active pharmaceutical ingredient [namely fipronil, (S)-methoprene, eprinomectin, and praziquantel] and their key degradation products in a broad-spectrum topical finished product. Typical sample of the finished product contains a total of 30-plus peaks of interest. Analytes were separated on a HALO C18 column (150 mm × 4.6 mm i.d., 2.7 µm particle size) with a gradient elution at 50 °C column temperature and 0.6 mL/min flow rate. Detection wavelength of 245 nm is used for praziquantel, eprinomectin and their degradation products, 265 nm for (S)-methoprene and its degradation products and 280 nm for fipronil and its degradation products and for the antioxidant, BHT. Mobile phase of the method is composed of 10 mM potassium phosphate buffer and 1,4- Dioxane (98/2, v/v, pH 5.0) as mobile phase-A, and EtOH/MeOH/MeCN/IPA (86/4/6/4, v/v/v/v) as mobile phase-B. All analytes of interest were adequately separated by this single HPLC method. The stability-indicating capability of the method has been demonstrated by successfully separating the degradation products in the stressed degraded samples of the finished product. Limit of quantitation (LOQ) and limit of detection (LOD) of the method is 0.3% and 0.1% of target analytical concentration for each individual API in the finished product. This method has been demonstrated to be sensitive, robust, specific, accurate and stability-indicating for analysis of the topical drug product containing praziquantel, fipronil, eprinomectin and (S)-methoprene.

Development and validation of a single HPLC method for the determination of thirteen pharmaceuticals in bulk and tablet dosage form

The aim of this study was to develop and validate a high performance liquid chromatography (HPLC) method for the determination of thirteen selected pharmaceutical compounds (metformin, amoxicillin, chloroquine, theophylline, trimethoprim, caffeine, norfloxacin, ciprofloxacin, acetylsalicylic acid, doxycycline hyclate, metronidazole, albendazole and cloxacillin) in bulk and tablet dosage form. Chromatographic separation using a Kromasil C18 column, gradient elution with aqueous formic acid (0.1%), methanol and acetonitrile, a UV absorption wavelength of 250 nm and a mobile phase flow rate of 1 mL/min over a 22 min run time was optimized for complete separation of the selected target compounds. The method was validated and results for: linearity, precision, sensitivity, accuracy, specificity, suitability and method robustness were obtained and met the ICH guidelines. Calibration curve correlation coefficients ranged from 0.9985-0.9998 and the percentage relative standard deviations for repeated analysis was below 5%, indicating acceptable method precision. The limits of detection (LODs) and quantification (LOQs) ranged from 0.020-0.27 µg/L and 0.080-0.91 µg/L, respectively. The accuracy study yielded recoveries in the ranges 86.0-102% for pure compounds and 90.9-106% for compounds in tablet dosage form. The method is robust for small or deliberate changes to the chromatographic parameters and found to be appropriate for analysis of tablets for the determination of the thirteen pharmaceuticals.
 
 KEY WORDS: Pharmaceuticals, Bulk determination, Tablet dosage, High performance liquid chromatography, Method development, ICH guidelines
 
 Bull. Chem. Soc. Ethiop. 2021, 35(1), 17-31.
 DOI: https://dx.doi.org/10.4314/bcse.v35i1.2

Development and Clinical Evaluation of a High-Throughput LC-MS/MS Assay for Vitamin B6 in Human Plasma and Serum.

Pyridoxal 5'-phosphate (PLP) is the primary circulatory form of vitamin B6, an essential cofactor for numerous biochemical enzymatic reactions. Conventional PLP analysis using high-performance liquid chromatography (HPLC) with fluorescence requires derivatization and long injection-to-injection time. Development of high-throughput LC-MS/MS assays is desirable. Stable isotope labeled internal standard was added to aliquots of samples, proteins were precipitated using trichloroacetic acid, and supernatants were analyzed by multiple reaction monitoring using LC-MS/MS in positive ion mode. Analysis time for PLP was 3.0 min using single column HPLC separation and 2.4 min using alternating column regeneration (ACR). Clinical evaluation of the method included review of results (n = 102 386) from routine performance of the assay. The assay was linear to 500 nmol/L; limit of quantification was 5 nmol/L. Imprecision (CV) of the assay was <5%. Equivalent performance was observed for single HPLC column and ACR. In 62% of routinely analyzed patient samples, PLP concentrations were within the reference interval; higher PLP concentrations were observed in samples from males than from females. Vitamin B6 deficiency was lowest in children and highest in elderly adults. Lower PLP concentrations were observed in samples collected during winter/spring than during summer/fall. We observed lower concentrations in plasma collected in lithium heparin tubes, suggesting PLP degradation caused by the anticoagulant. This LC-MS/MS method allows PLP determination using simple sample preparation and short analysis time. We observed association of PLP concentrations with age, sex, and season of sample collection. Our data indicate that lithium heparin anticoagulant tubes reduce measured PLP concentration.

Development and Validation of a Single HPLC Method for Analysis of Purines in Fish Oil Supplements

Gout is one of the most common forms of inflammatory arthritis, affecting over 8 million adults in the US. Individuals with gout are advised to avoid the habitual intake of purine-rich foods such as meats, seafood, purine-rich vegetables, and animal protein. An increased risk of developing or having subsequent attacks of gout is particularly associated with the consumption of seafood. However, clinical studies have shown that certain seafood and fish oil supplements that contain large amounts of omega-3 fatty acids provide important cardiovascular benefits. Individuals who might benefit from omega-3 fatty acid supplementation may therefore avoid fish oil products because they contain purines. Currently, there are no distinct high-performance liquid chromatography (HPLC) methods available in the literature that are validated as per the International Conference on Harmonisation (ICH) guidelines for the analysis of omega-3 fatty acid oils or fish oil containing products for purine content. A robust, fast, and efficient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the specific analysis of the naturally occurring purines guanine, purine, theobromine, and adenine. These purines are often found in fish oil and seafood. The analytical method reported herein quantifies all four purines in fish oil in about 20 minutes.

A single extraction and HPLC procedure for simultaneous analysis of phytosterols, tocopherols and lutein in soybeans

A saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for simultaneous analysis of phytosterols, tocopherols and lutein (a carotenoid) in soybeans. Separation was achieved on a phenyl column with a ternary, isocratic solvent system of acetonitrile, methanol and water (48:22.5:29.5, v/v/v). Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol, stigmasterol, campesterol, and α-, δ- and γ-tocopherols, while lutein was quantified with visible light absorption at 450nm. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (R2>0.99) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. Also, the accuracy of results of four soybeans using the described saponification and HPLC analytical method was validated against existing methods. This method offers a more efficient alternative to individual methods for quantifying lutein, tocopherols and sterols in soybeans.

HPLC method development and validation for simultaneous detection of Arabinoside-C and doxorubicin

This paper describes a new validated high performance liquid chromatography (HPLC) method for the simultaneous determination of two anti-cancer drugs, Arabinoside-C (Ara-C) and doxorubicin hydrochloride (DOX). A simultaneous determination method saves cost and time as both drugs can be injected into a single HPLC system without the need to change or re-equilibrate with a new mobile phase. The objective of the study is to develop a simultaneous determination method of two anti-cancer drugs, Ara-C and DOX. The mobile phase consisted of a mixture (45:55) of acetonitrile:ammonium hydrogen phosphate aqueous solution (0.01 M) at pH 6.2 at a flow rate of 0.3 ml/min, with UV detection at 252 nm. Separation was achieved on a C-18 column (5 µm: 250 mm × 4.6 mm) maintained at 30°C in a column oven. The method was linear between 325 ng/ml and 10 µg/ml for Ara-C and 625 ng/ml and 20 µg/ml for DOX. The limit of detection (LOD) was 20 ng/ml for Ara-C and 60 ng/ml for DOX. The developed HPLC method achieved good precision and accuracy as well as limit of quantitations. The developed and validated method is suitable to be used for routine analysis of Ara-C and DOX.

An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP–glucose:tetrahydrobiopterin glucosyltransferase

The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular disorders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP–glucose α-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP–glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin–glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4.

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.

Absence of correlation between glycated hemoglobin and lipid composition of erythrocyte membrane in type 2 diabetic patients

Correlation of glycated hemoglobin (HbA 1c) level with degrees of certain peroxidative changes in erythrocyte membrane lipids in diabetic patients have been reported. In the present study, peroxidation of erythrocyte lipids was assessed by changes in tocopherols (Toc), phospholipids (PL), and malondialdehyde (MDA). Membrane cholesterol, Toc, and PL were determined from the same lipid extract. Toc and cholesterol were measured simultaneously by high-performance liquid chromatography (HPLC), and each PL class was determined by a single HPLC elution with ultraviolet light (UV) detection. The detection of PL with UV depends primarily on double bonds in fatty acids and shows a decrease in fatty acids by peroxidation. Changes in Toc and each PL were calculated on the basis of cholesterol and SM, respectively, since cholesterol and sphingomyelin (SM) in the cell membrane are not prone to peroxidation. MDA was measured by an HPLC method with fluorescence detection. These methods for assessment for peroxidation of membrane lipids in intact erythrocytes were validated by experiments with 2, 2-azobis(2-amidinopropane)dihydrochloride (AAPH) and tert-butylhydroperoxide (tBHP); nevertheless, significant differences in the levels of Toc, each PL class, and MDA between a high-HbA 1c group and a low-HbA 1c group were not detected.

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography–tandem mass spectrometry

Direct plasma injection technology coupled with a LC–MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within ±15%. The advantages and challenges of using direct single column LC–MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.

Analytical Procedure for Simultaneous Use of Seven Fluorobenzoates in Multitracer Tests

An analytical procedure allowing simultaneous use of seven fluorobenzoate (FBA) isomers was developed and examined through column and field tracer experiments. The analytical method, based on high performance liquid chromatography (HPLC), included a reverse-phase separation method. This method was found to be very efficient for the FBA analysis, allowing accurate determination of seven FBAs in a single HPLC run, while avoiding analytical interference of other natural water constituents or pollutants. Consequently, this separation method allows the simultaneous use of seven isomers in multitracer tests in highly saline or contaminated environments. The mobility properties of the FBAs were examined in percolation experiments through a column filled with a mixture of crushed chalk and common fracture filling and coating material. The FBAs behaved conservatively as compared to bromid. All seven FBA isomers were successfully used in a multitracer test in a field percolation experiment designed to identify flowpath along a fracture plane.

High-Capacity Screening of Arylalkylamine N-Acetyltransferase Inhibitors using a High-Performance Liquid Chromatography System

Systematic screening is a natural development of any pharmacological program. Most enzyme inhibitor screens use indirect or “aspecific” methods, such as colorimetric or fluorimetric ones. These screening methods cause quite a few false-positive and false-negative hits. In order to limit these as much as possible, we developed a methodology using a high-performance liquid chromatography (HPLC) system for the medium throughput screening of serotonin N-acetyltransferase inhibitors. The core of this screening system is (1) the dramatic shortening of the analytical time down to 100 s per run by using a high-performance analytical column (Turbo), and (2) the use of absorption as opposed to radioactivity for detection of the product of the reaction (N-acetylserotonin). This system permits the analysis of about 1,000 compounds per day to be performed with a single HPLC system. This enzymatic system was taken as an example, because the methodology can be extended to many other enzymes, particularly transferases, phosphatases, and kinases.

An Improved Method for the Determination of Green and Black Tea Polyphenols in Biomatrices by High-Performance Liquid Chromatography with Coulometric Array Detection

Tea polyphenols are strong antioxidants and are believed to have beneficial health effects. However, the blood and tissue levels of these compounds are not well characterized because of a lack of suitable analytical methods for the biological resolution of these compounds. Previously, we developed methods for the analysis of three green tea catechins. Now we report an improved method for the measurement of the levels of the different catechins and theaflavins in biological fluids and tissues. The method includes digestion of the plasma, urine, or tissue samples with β-d-glucuronidase and sulfatase, followed by extraction with ethyl acetate and subsequent separation by reversed-phase high-performance liquid chromatography (HPLC). The polyphenols are identified on the basis of their retention times, spectral analysis, and electrochemical behavior across an array of electrodes. In a single HPLC run, it is possible to determine the major catechins and theaflavins as well as some of the catechin metabolites. The detection limits for catechins and theaflavins are from 5 to 10 ng/ml of saliva, plasma, or urine.

A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydr oxyeicosatrienoic acid (5,6-DHT) to 5,6-δ-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-δ-lactone and 5,6-DHT with a t1/2 ≈ 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (λ = 205) comigrating with 5,6-δ-lactone. Incubation of 5,6-δ-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 ± 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 ± 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 ± 5.5 pg/ml and 21.6 ± 6.3 pg/ml, respectively, by arachidonic acid.—Fulton, D., J. R. Falck, J. C. McGiff, M. A. Carroll, and J. Quilley. A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol.

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.

Release of endogenous glutamate and γ-aminobutyric acid from rat striatal tissue slices measured by an improved method of high performance liquid chromatography with electrochemical detection

The release of endogenous glutamate and γ-aminobutyric acid (GABA) from rat brain tissue slices was studied using a tissue slice assay in which detectable amounts of the amino acids were released from 1–2 mg of tissue. An improved method of high performance liquid chromatography (HPLC) with electrochemical detection was employed to measure both glutamate and GABA after derivatization with o-phthalaldehyde and sulfide in a single isocratic HPLC analysis. The non-endogenous amino acid, homoglutamine, was used as an internal standard in verifying the consistent derivatization of amino acids and in quantifying amounts of glutamate and GABA released from the caudate-putamen tissue. The derivatized amino acids (1–30 pmol) were detected as chromatographic peaks eluting at baseline level and free of significant interfering co-eluates in a 25–30 min analysis time.

Simultaneous liquid chromatographic determination of vitamins A, E and β-carotene in common dairy foods

Milkfat was extracted quantitatively from dairy foods and the vitamins A, E and beta-carotene in the extract were analysed by normal phase high performance liquid chromatography (HPLC) using a silica column, a fluorescence detector and a UV/visible detector. Products such as butter and anhydrous milkfat were dissolved simply in hexane, whereas milk and milk powders underwent a Rose-Gottlieb fat extraction prior to HPLC analysis. The extraction method used as superior to commonly used saponification procedures in terms of speed as well as reduced loss and isomerization of the vitamin components in the food, because the chemical form of the vitamin is not changed to a less stable form such as retinol during extraction. The individual geometrical isomers of vitamin A, which possess different in vivo vitamin activities, were uniquely identified and quantified. Recovery studies showed satisfactory results for all three vitamins. The simultaneous nature of the assay allowed the estimation of the total vitamin A activity of the food because both vitamin A and pro-vitamin A (beta-carotene) were quantified. The single extraction and single HPLC analysis used for the three vitamins made the method well suited for routine work. (C) 1996 Elsevier Science Limited

Systematic analysis of glucoiridoids from Penstemon serrulatus Menz. by high-performance liquid chromatography with pre-column solid-phase extraction

Samples (122) of crude ethanol-extracts of callus tissues from Penstemon serrulatus Menz. were used to develope a solid-phase extraction (SPE) clean-up procedure using the octadecylsilica packed cartridge for removing a complex mixture of free phenolic acids and anthocyanine-like colour substances, for the isolation of a sufficiently clean glucoiridoid fraction (GF). An additional SPE sample pretreatment step of the crude extracts enabled the enhancement of selectivity and sensitivity on applied high-performance liquid chromatography (HPLC) for the identification and quantitation of the iridoid constituents of the GF fraction. In particular, the presence of five valeriana-type esterified glucoiridoids which consist of penstemide, serrulatoloside, 8-epi-valerosidate, 7-desoxy-8-epi-valerosidate and serrulatoside in prepared GF fractions were determined by isocratic HPLC measurements. During a single HPLC separation, the traces of four non-esterified glucoiridoids, i.e. harpagide, aucuboside, loganine and plantarenaloside, were satisfactorily resolved and detected on the registered HPLC chromatograms of investigated GF fractions. The HPLC analyses were carried out on an octadecylsilica column (25 × 0.4 cm I.D.) using methanol-water (30:7070, v/v) as the mobile phase with a flow-rate of 2 ml/min and the absorbance was monitored at 220 nm using an UV detector. The described chromatographic assay for penstemide, which exhibits potential antitumor activity against the P-388 lymphocytic leukemia cells, was applied to the monitoring and standardization of growth conditions for the callus cultures of P. serrulatus. Penstemide contents were found to range from 0.05% to 2.7% of the fresh weight of the investigated callus samples. Multivariate statistical methods (principal components analysis) was applied to demonstrate the influence of a variety of compositions of growth media (especially the type and concentration of synthetic growth regulators, e.g. 3-indolylacetic acid, 2,4-dichlorophenoxyacetic acid or 1-naphthylacetic acid, on the formation of different profiles of glucoiridoids in the callus cultures of P. serrulatus. Calculated principal component values were found to be useful for the explanation of variations in the penstemide/serrulatoloside ratio in the investigated samples and for determining the most favourable growth conditions in plants leading to optimal glucoiridoid biosynthesis.