[Determination of multiple residual solvents in ibandronate sodium using headspace-gas chromatography].

Ibandronate sodium, a third-generation diphosphate drug used worldwide to treat osteoporosis, has the advantages of convenient use, low toxicity, and significant therapeutic effects. However, the residual organic solvents in the synthesis process of sodium ibandronate not only have a negative impact on the efficacy of the drug, but also lead to a decrease in drug stability. Moreover, if the residual amounts of these solvents exceed safety standards, they may pose serious threats to human health. This study successfully established a convenient and efficient method based on headspace-gas chromatography (HS-GC) for the simultaneous determination of five residual solvents (methanol, acetone, benzene, toluene, 1-pentanol) in the raw materials of ibandronate sodium. The results indicated that satisfactory analytical performance can be achieved by using DB-624 capillary column (30 m×0.32 mm×1.8 µm) and a flame ionization detector in conjunction with headspace autosampling and a temperature program. The specific operating conditions included an initial temperature of 40 ℃, with a hold of 2 min, followed by a temperature ramp first to 200 ℃ at a rate of 5 ℃/min and then to 240 ℃ at a rate of 20 ℃/min, with a hold of 5 min. Nitrogen with a flow rate of 1 mL/min and split ratio of 14∶1 was used as the carrier gas. The headspace vial temperature was maintained at 80 ℃, and the sample equilibration time was 20 min. Under the established analytical conditions, good linear relationships were obtained between the mass concentrations of methanol (72-216 µg/mL), acetone (120-360 µg/mL), benzene (0.048-0.144 µg/mL), toluene (21.36-64.08 µg/mL), and 1-pentanol (120-360 µg/mL) and their corresponding peak areas, with correlation coefficients (r) greater than 0.990. The limits of detection for these solvents were 2.88, 0.011, 0.90, 0.24, and 0.024 ng/mL, respectively, with limits of quantification of 11.5, 0.043, 3.6, 0.96, and 0.096 ng/mL, respectively. Furthermore, the recoveries of these solvents ranged from 86.3% to 101.9%, with relative standard deviations (RSDs, n=3) of less than 2.49%. The proposed method is simple, accurate, reliable, and suitable for the rapid and simultaneous determination of five residual solvents in the raw materials of ibandronate sodium. This study has important practical significance in improving drug safety and ensuring public health.

Comparative Evaluation of the Effect of Different Chelating Agents on Mineral Content and Erosion of Radicular Dentine: A FESEM-EDS Analysis.

OBJECTIVE: Irrigating solutions play an important role in the debridement and disinfection of the root canal space, and thus, it is crucial to comprehend their effects on the composition and surface structure of radicular dentine. This study evaluated and compared the effects of 17% ethylenediaminetetraacetic acid (EDTA), 9% 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and 0.2% chitosan on the mineral content and erosion of radicular dentine when used as a final rinse. METHODS: Sixty extracted human mandibular premolar teeth were decoronated and instrumented to ProTaper size F2. After final instrumentation, the samples were randomly divided into 4 groups (n=15) according to the type of final irrigant used: Normal saline (control), 17% EDTA, 9% HEBP and 0.2% chitosan. Field emission scanning electron microscopy was used to assess the erosion of radicular dentine, and energy dispersive X- ray spectroscopy was used to quantify the radicular dentine mineral composition at the coronal, middle and apical levels of all the prepared samples after final irrigation. The one-way analysis of variance was used for intra-group and inter-group comparisons of means, the Kruskal Wallis test for intra-group and inter-group comparisons of medians and Tukey's post hoc test for pairwise comparisons. RESULTS: There was no significant difference in the levels of calcium (Ca), phosphorus (P) and Ca/P ratio after final rinse with 17% EDTA, 9% HEBP and 0.2% chitosan at all three root levels (p>0.05); except at the coronal level, where 0.2% chitosan caused significantly less alteration in Ca levels and Ca/P ratio than 17% EDTA and 9% HEBP respectively (p<0.05). 17% EDTA, 9% HEBP and 0.2% chitosan caused no erosion at the middle and apical levels. Meanwhile, 17% EDTA and 9% HEBP caused moderate erosion at the coronal level. CONCLUSION: Alternatives to 17% EDTA during final irrigation can be 9% HEBP and 0.2% chitosan. (EEJ-2023-06-078).

Long-term monitoring of clodronate in equine hair using liquid chromatography-tandem mass spectrometry.

Given the potential for long-term inhibition of bone remodeling/healing and detrimental effects to horses in training, bisphosphonates are tightly regulated in horseracing. Hair has proven to be an effective matrix for detection of drug administration to horses and has been particularly effective in detecting drugs for a long period of time post administration. Thus, hair may prove to be a useful matrix for detection of administration of this class of drugs. The objective of the current study was to develop an assay and assess the usefulness of hair as a matrix for long-term detection of clodronate to horses. Seven horses received a single intramuscular administration of 1.8 mg/kg clodronate. Hair samples were collected prior to and up to 6 months post administration. A liquid chromatography-tandem mass spectrometry method was developed and concentrations of clodronate measured in hair samples. The drug was first detected on day 7 in 4/7 horses, and on days 14, 28 and 35 in the remaining three horses. In 4/7 horses, clodronate was still detectable 6 months post administration. Results of this study demonstrate that, although there was significant inter-individual variability in detection times (63 to 180 days) and several intermediate times where the drug could not be detected but was subsequently detected in later timepoints, clodronate administration was detectable in hair for a prolonged period in most of the horses (4/7) studied.

Molecular recognition of bisphosphonate-based drugs by di-zinc receptors in aqueous solution and on gold nanoparticles.

Metal-based anion receptors have several important applications in sensing, separation and transport of negatively charged species. Amongst these receptors, di-zinc(ii) complexes are of particular interest for the recognition of oxoanions, in particular phosphate derivatives. Herein we report the synthesis of a di-zinc(ii) receptor and show that it has high affinity and selectivity for bisphosphonates such as alendronate and etidronate - which are used to treat a number of skeletal disorders as well as showing interesting anticancer properties. The binding mode of the di-zinc(ii) receptor with alendronate and etidronate has been unambiguously established by single crystal X-ray crystallography. In addition, by modifying the backbone of the receptor, we show that the drug-loaded receptor can be attached onto gold nanoparticles as potential drug-delivery vehicles.

Absolute radiotracer concentration measurement using whole-body solid-state SPECT/CT technology: in vivo/in vitro validation.

The accuracy of recently approved quantitative clinical software was determined by comparing in vivo/in vitro measurements for a solid-state cadmium-zinc-telluride SPECT/CT (single photon emission computed tomography/x-ray computed tomography) camera. Bone SPECT/CT, including the pelvic region in the field of view, was performed on 16 patients using technetium-99m methylene diphosphonic acid as a radiotracer. After imaging, urine samples from each patient provided for the measurement of in vitro radiopharmaceutical concentrations. From the SPECT/CT images, three users measured in vivo radiotracer concentration and standardized uptake value (SUV) for the bladder using quantitative software (Q.Metrix, GE Healthcare). Linear regression was used to validate any in vivo/in vitro identity relations (ideally slope = 1, intercept = 0), within a 95% confidence interval (CI). Thirteen in vivo/in vitro pairs were available for further analysis, after rejecting two as clinically irrelevant (SUVs > 100 g/mL) and one as an outlier (via Cook's distance calculations). All linear regressions (R2 ≥ 0.85, P < 0.0001) provided identity in vivo/in vitro relations (95% CI), with SUV averages from all users giving a slope of 0.99 ± 0.25 and intercept of 0.14 ± 5.15 g/mL. The average in vivo/in vitro residual difference was < 20%. Solid-state SPECT/CT imaging can reliably provide in vivo urinary bladder radiotracer concentrations within approximately 20% accuracy. This practical, non-invasive, in vivo quantitation method can potentially improve diagnosis and assessment of response to treatment. Graphical abstract.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Localization of Minodronate in Mouse Femora Through Isotope Microscopy.

Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate's effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([(15)N]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [(15)N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [(15)N]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase-positive osteoclasts and alkaline phosphatase-reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.

Simultaneous determination of two amino bisphosphonates drugs by micellar electrokinetic chromatography.

A sensitive micellar electrokinetic chromatographic method with laser-induced fluorescence detection was developed for the determination of alendronate sodium (ALN) and pamidronate disodium (PAM) after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The developed method was first used for the determination of ALN and PAM in pharmaceutical preparations. After optimization, baseline separation of the analytes could be obtained in <10 min in a running buffer composed of 10 mM sodium borate and 30 mM sodium dodecyl sulfate (pH 9.0) at a voltage of 15 kV with 25°C cartridge temperature and the samples were injected by pressure (3447.5 Pa × 3 s). The method has linearity range of 0.05-70 µg/mL for the analytes (correlation coefficients: 0.9995 and 0.9997), the detection limits were 4 and 10 ng/mL for ALN and PAM, respectively. In intraday precision experiment, the relative standard deviation (RSD) values for migration time were 0.30% (ALN) and 0.27% (PAM), and the RSD values for peak areas were 1.19% (ALN) and 1.32% (PAM). The ranges of recovery were 95.5-101.8 and 94.6-105.3% for ALN and PAM, respectively. This method is not only rapid and accurate but also has the potential to be used for the quality control in pharmaceutical preparations of the two drugs.

On-cartridge derivatization coupled with solid-phase extraction for the ultra-sensitive determination of minodronic acid in human plasma by LC-MS/MS method.

Minodronic acid (MA) is a third-generation bisphosphonate (BP). Its high potency allows lower doses to be administered in clinical settings compared with other BPs, which results in extremely low systemic exposure. Therefore, it is essential to develop an ultra-sensitive bioassay for pharmacokinetics studies of MA. In this work, we used on-cartridge derivatization of MA with trimethylsilyldiazomethane to extract MA from plasma samples and improve its LC-MS/MS behavior. The reaction produced a known derivative, tetramethylated MA, and a novel derivative, pentamethylated MA (PMMA). PMMA exhibited a better signal-to-noise ratio, and was monitored for the quantification of MA. However, the derivatization yield of d4-PMMA was much lower and more variable than that of PMMA, which decreased the effectiveness of its correction function as an internal standard. Therefore, a two-cycle derivatization approach was introduced to increase its yield and improve the reproducibility. The calibration curves of MA showed good linearity over the range of 10.0-1000 pg/mL. A lower limit of quantification of 10.0 pg/mL was achieved with acceptable precision (<10.5%) and accuracy (5.0%). The intra- and inter-batch precision of quality control samples was <9.5%, and the accuracy ranged from -2.8% to 0.6%. The stability results showed that MA was stable in human plasma for 6h at room temperature (25°C), for 115 days at -20°C, during three freeze/thaw cycles (from -20°C to 25°C), and in post-preparative samples for 24h at 4°C. The method was successfully used to characterize the pharmacokinetic profile of MA following an oral dose of 1.0mg MA hydrate to healthy volunteers (n=12). The proposed derivatization procedure was also extended to measure other BPs (risedronic acid and zoledronic acid) in human plasma at low pg/mL.

Impurity profiling of ibandronate sodium by HPLC-CAD.

The modern bisphosphonate drug ibandronate sodium, a challenging candidate for impurity profiling, was analyzed using high performance liquid chromatography (HPLC) combined with corona charged aerosol detection (CAD). Separation was achieved on a mixed mode column combining hydrophobic C18 and strong anion exchange retention mechanisms using a mass spectrometer compatible volatile mobile phase consisting of trifluoroacetic acid and acetonitrile while gradient elution was applied. The method was validated following the ICH guideline Q2(R1) and found suitable for the assessment of ibandronate's related substances. The observed CAD-response for all identified impurities was linear (R(2)>0.995) over a small concentration range (0.05-0.25) and a quantification limit of at least 0.03% was found. Four batches of two different manufacturers were tested by means of the method. None of the batches contained a single impurity above 0.05%. The major impurities of all batches were the synthesis by-products N-desmethyl- and N-despentyl ibandronate as well as N,N-dimethyl pamidronate.

Quantitative analysis of bisphosphonates in biological samples.

Bisphosphonate drugs pose significant challenges for bioanalysis due to various complicating factors. In 2006, a novel approach, utilizing 'on-column' derivatization with diazomethane, was reported that revolutionized the application of liquid-chromatography-tandem mass spectrometry to bisphosphonates bioanalysis. The methodology enables superior biological sample clean-up while transforming bisphosphonates into species amenable to liquid-chromatography-tandem mass spectrometry detection. Since then, the approach has been successfully applied to numerous bisphosphonates. The use of an alternative methylation reagent - trimethylsilyl diazomethane - for on-column derivatization has been reported recently. This review focuses on published methods utilizing on-column derivatization for bioanalysis of major bisphosphonate drugs in biological matrices. Critical points required for successful application of on-column derivatization to the bioanalysis of bisphosphonates will be discussed.

Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology.

A fast and reliable nuclear magnetic resonance spectroscopic method for quantitative determination (qNMR) of targeted molecules in reference materials has been established using the ERETIC2 methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). The developed approach was validated for the analysis of pharmaceutical samples in the context of official medicines control, including ibandronic acid, amantadine, ambroxol and lercanidipine. The PULCON recoveries were above 94.3% and coefficients of variation (CVs) obtained by quantification of different targeted resonances ranged between 0.7% and 2.8%, demonstrating that the qNMR method is a precise tool for rapid quantification (approximately 15min) of reference materials and medicinal products. Generally, the values were within specification (certified values) provided by the manufactures. The results were in agreement with NMR quantification using an internal standard and validated reference HPLC analysis. The PULCON method was found to be a practical alternative with competitive precision and accuracy to the classical internal reference method and it proved to be applicable to different solvent conditions. The method can be recommended for routine use in medicines control laboratories, especially when the availability and costs of reference compounds are problematic.

Quantitation of zoledronic acid in murine bone by liquid chromatography coupled with tandem mass spectrometry.

An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250-50.0µg/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06±0.52 vs. 0.76±0.09ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment.

Tracking bisphosphonates through a 20 mm thick porcine tissue by using surface-enhanced spatially offset Raman spectroscopy.

Track it down: A recognized surface-enhanced Raman scattering (SERS) nanotag signal was monitored from a thin, dispersed layer of bisphosphonate-functionalized nanotags on a bone sample, through a 20 mm thick specimen of porcine muscle tissue by surface-enhanced spatial offset Raman spectroscopy (SESORS; see picture). The result demonstrates the great potential for non-invasive in vivo bisphosphonate drug tracking.

Assessment of a bone biopsy technique for measuring tiludronate in horses: a preliminary study.

This study assessed the feasibility of measuring tiludronate in horses using a minimally invasive bone biopsy technique. Eight horses were treated with intravenous (IV) tiludronate [1 mg/kg bodyweight (BW)], either once (n = 4) or twice, 28 d apart (n = 4). The horses that were treated once were euthanized on days 1, 43, 57, or 92 and those that were treated twice, were euthanized on days 112, 154, 194, or 364. Bone samples were taken bilaterally from each horse at 4 sites: the third metacarpal bone (MCIII), the 13th rib (R13), the tuber coxae (TC), and the cuboid bone (CB). Test samples were taken with a 5-mm diameter dental drill, while larger reference samples were taken with an osteotome. The concentrations of tiludronate were measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The TC was the easiest site to sample, and no technical difficulties were encountered for extraction and measurement. Drill sampling at the MCIII was difficult. Moreover, both the extraction and measurement caused technical problems and results were unreliable in most cases (93%). Drill samples obtained from the R13 were very small and access to the CB required considerable dissection, which would not be feasible in vivo. Forty-six percent and 36% of the tiludronate measurements performed on the R13 and CB samples, respectively, were unreliable. The ratio of tiludronate concentrations ranged from 73% to 185% (median: 118%) in the TC, 65% to 208% (median: 81%) in the R13, and 26% to 110% (median: 57%) in the CB. In all but 1 horse, the highest concentrations of tiludronate were found in the TC. It was concluded that bone biopsies performed at the TC were adequate for measuring tiludronate in horses and should be considered in future for repeated measurements over time in living animals.

Capillary electrophoresis-electrochemiluminescence detection method for the analysis of ibandronate in drug formulations and human urine.

A simple, rapid and sensitive CE method coupled with electrochemiluminescence (ECL) detection for direct analysis of ibandronate (IBAN) has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 9.0) and a voltage of 13.5 kV, separation of IBAN in a 30-cm length capillary was achieved in 3 min. ECL detection was performed with an indium tin oxide working electrode bias at 1.6 V (versus a Pt wire reference) in a 200-mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)(3)(2+) (where bpy=2,2'-bipyridyl). Derivatization of IBAN prior to CE-ECL analysis was not needed. Linear correlation (r=0.9992, n=7) between ECL intensity and analyte concentration was obtained in the range of 0.25-50 µM IBAN. The LOD of IBAN in water was 0.08 µM. The developed method was applied to the analysis of IBAN in a drug formulation and human urine sample. SPE using magnetic Fe(3)O(4)@Al(2)O(3) nanoparticles as the extraction phase was employed to pretreat the urine sample before CE-ECL analysis. The linear range was 0.2-12.0 µM IBAN in human urine (r=0.9974, n=6). The LOD of IBAN in urine was 0.06 µM. Total analysis time including sample preparation was <1 h.

Stability indicating ion chromatography method for the simultaneous determination of ibandronate sodium drug substance and its impurities.

A simple and sensitive ion chromatography method has been developed for the simultaneous assay of ibandronate sodium drug substance and the determination of its impurities. The separation was achieved on Allsep™ anion column 150 mm × 4.6 mm, 7 µm particle diameter. The mobile phase consisted of 1% (v/v) aqueous formic acid and acetone 98:2% (v/v); flow rate 1.0 ml min(-1) at ambient temperature. The analytes were monitored by conductometric detector. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolytic, thermal and humidity degradation. Considerable degradation was achieved only under oxidative conditions. Mass balance was demonstrated in all stress conditions. The method was validated for specificity, precision, linearity, solution stability and accuracy. The limits of detection (LOD) and limits of quantification (LOQ) for impurities were in the range of 0.36-0.80 µg ml(-1) and 1.00-2.40 µg ml(-1), respectively. For ibandronate LOD was 38 µg ml(-1) and LOQ was 113 µg ml(-1). The average recoveries for impurities and ibandronate were in the range of 99.0-103.1% and the method can be successfully applied for the routine analysis of ibandronate sodium drug substance.

Nuclear magnetic resonance-based quantification of organic diphosphates.

Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogs have been prepared for subsequent evaluation. Prior to biological experiments, it is typically necessary to determine the concentration of the target molecule in solution. Here we describe a method where concentrations of stock solutions of organic diphosphates and bisphosphonates are quantified using (31)P nuclear magnetic resonance (NMR) spectroscopy with standard instrumentation using a capillary tube with a secondary standard. The method is specific and is applicable down to a concentration of 200 µM. The capillary tube provides the reference peak for quantification and deuterated solvent for locking.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Rapid analysis of alkylphosphonate drugs by capillary zone electrophoresis using indirect ultraviolet detection.

A rapid capillary electrophoretic method for the analysis of three alkylphosphonate drugs (i.e. fosfomycin disodium (FOS), clodronate disodium (CLO) and alendronate sodium (ALN)) was developed by using multiple probe BGE and indirect UV detection. BGE containing 30 mM benzoic acid, 5 mM salicylic acid and 0.5 mM CTAB (pH 3.8), temperature of 30 degrees C, applied voltage of -30 kV and detection at 220 nm provided baseline separation of all analytes (resolution (R)>2.2) in 3.2 min. EOF reversal by addition of CTAB and negative voltage polarity leading to the co-EOF flow and short analysis time. Two probe BGE greatly improved peak symmetry. The method showed good linearity (r(2)>0.999 in ranges of 20-1000 microg/mL for FOS, 100-1000 microg/mL for CLO and 100-750 microg/mL for ALN) repeatablitiy (RSD<2.15%), recovery (99.3-101.1%) and sensitivity (LOD<50 microg/mL). Freshly prepared BGE and sample solutions are essential for the method precision and accuracy. This new method can be utilized for routine analysis of FOS, CLO and ALN in dosage forms because of its efficiency, reliability, speed and simplicity.