Validated spectrofluorimetric assay of two co-administered drug mixtures containing hydroxychloroquine with either moxifloxacin or ofloxacin as a drug regimen for hospital-acquired pneumonia in patients with COVID-19

Moxifloxacin and ofloxacin are two broad-spectrum quinolone antibiotics. They are among the most widely used antibiotics, at this time, applied to control the COVID-19 pandemic. Hydroxychloroquine is an FDA-approved drug for the treatment of COVID-19. This work describes a simple, green, selective, and sensitive spectrofluorimetric method for the assay of moxifloxacin and ofloxacin in the presence of hydroxychloroquine, two co-administered mixtures used in the treatment of hospital-acquired pneumonia in patients with COVID-19. Simultaneous assay of hydroxychloroquine and moxifloxacin was carried out in methanol using a direct spectrofluorimetric method (method I) at 375 and 550 nm, respectively, after excitation at 300 nm. The direct spectrofluorimetric assay was rectilinear over concentration ranges 50.0–400.0 and 300.0–2500.0 ng/ml for hydroxychloroquine and moxifloxacin, respectively, with limits of detection (LOD) of 6.4 and 33.64 ng/ml and limits of quantitation (LOQ) of 19.4 and 102.6 ng/ml, respectively, for the two drugs. The assay for hydroxychloroquine and ofloxacin was carried out by measuring the first derivative synchronous amplitude for hydroxychloroquine at the zero crossing point of ofloxacin and vice versa at Δλ = 140 nm (method II). Hydroxychloroquine was measured at 266 nm, while ofloxacin was measured at 340 nm over the concentration range 4–40 ng/ml for hydroxychloroquine and 200–2000 ng/ml for ofloxacin with LOD of 0.467 and 25.3 ng/ml and LOQ of 1.42 and 76.6 ng/ml, respectively, for the two drugs. The two methods were validated following International Conference on Harmonization guidelines and were applied to the analysis of the two drugs in plasma with good percentage recoveries (109.73–93.17%).     

A highly sensitive micelle-enhanced synchronous spectrofluorimetric determination of the recently approved co-formulated drugs,bilastine and montelukast in pharmaceuticals and human plasma at nanogram levels

In this study, the simultaneous determination of bilastine and montelukast, two recently approved co-formulated antihistaminic medications, was accomplished using a quick, sensitive, environmentally friendly, and reasonably priced synchronous fluorescence spectroscopic approach for the first time. Enhancement of the method's sensitivity down to nanogram levels was achieved by the addition of sodium dodecyl sulfate (1.0% w/v) as a micellar system. According to the results, bilastine and montelukast's fluorescence was measured at 255.3 and 355.3 nm, respectively, using Δλ of 40.0 nm and distilled water as a green diluting solvent. With respect to the concentration ranges of bilastine (5.0–300.0 ng/ml) and montelukast (50.0–1000.0 ng/ml), the method showed excellent linearity (r ≥ 0.9998). The results showed that the suggested method is highly sensitive, with detection limits of 1.42 and 13.74 ng/ml for bilastine and montelukast, respectively. Within-run precisions (intra- and interday) per cent relative standard deviations (RSD) for both analytes were <0.59%. With high percentage recoveries and low percentage RSD values, the designed approach was successfully applied for the simultaneous estimation of the cited medications in their dosage form and human plasma samples. To evaluate the green profile of the suggested method, an analytical GREENNESS metric approach (AGREE) and green analytical procedure index (GAPI) metric tools were used. These two methods for evaluating greenness confirmed that the developed method met the highest number of green requirements, recommending its use as a green substitute for the routine analysis of the studied drugs. The proposed approach was validated according to ICHQ2 (R1) guidelines.     

Simultaneous determination of amlodipine and metoprolol in their combined dosage form using a synchronous fluorescence spectrofluorimetric method

Highly sensitive, rapid, accurate and precise synchronous fluorescence spectrofluorimetric method has been developed for simultaneous analysis of a mixture of amlodipine (AMD) and metoprolol (MET). The method relies on measuring the relative synchronous fluorescence intensity of both drugs at Δλ of 90 nm in acetate buffer solution at pH 5. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.2–2 μg/ml and 0.5–10 μg/ml for AMD and MET, respectively. The limits of detection were 50 ng/ml for AMD and 130 ng/ml for MET while the limits of quantitation were 150 ng/ml for AMD and 390 ng/ml for MET. The developed method was applied successfully for the determination of the two drugs in their co‐formulated tablet. The mean percent recoveries were found to be 100.51 and 99.57 for AMD and MET, respectively.     

First derivative synchronous spectrofluorimetric method for the simultaneous determination of propofol and cisatracurium besylate in biological fluids

Propofol and cisatracurium besylate have been simultaneously determined using a highly sensitive first derivative synchronous spectrofluorometric method. The method is based on measuring first derivative synchronous spectrofluorimetric amplitude at Δλ = 40 nm with a scanning rate of 600 nm/min. The different experimental parameters affecting the fluorescence intensity of the two drugs were carefully studied and optimized. The amplitude–concentration plots were rectilinear over the range 40.0–400.0 ng/mL and 20.0–280.0 ng/mL for propofol and cisatracurium, respectively with lower detection limits of 4.0 and 2.35 ng/mL and quantification limits of 12.1 and 7.1 ng/mL for propofol and cisatracurium, respectively. The proposed method was successfully applied for the determination of the two compounds in synthetic mixtures and in commercial ampoules. The high sensitivity attained using the proposed method allowed the simultaneous determination of both drugs in spiked plasma samples. The mean % recoveries in spiked human plasma (n = 3) were 96.53 ± 0.90 and 96.20 ± 1.64 for each of propofol and cisatracurium, respectively. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.     

Eco-friendly synchronous fluorescence spectrofluorimetric method for simultaneous determination of montelukast sodium and fexofenadine hydrochloride in their antiallergic rhinitis fixed-dose combination tablets

An innovative, simple, accurate, sensitive, and eco-friendly synchronous fluorescence spectrofluorimetric method has been developed for the simultaneous analysis of montelukast sodium (MON) and fexofenadine hydrochloride (FEX). The method relies on measuring the relative synchronous fluorescence intensity of both drugs using Δλ of 60 nm in methanol at 405 nm for MON and 288 nm for FEX. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.1–2.0 and 2.0–20.0 μg/ml for MON and FEX, respectively. The limits of detection were 0.018 and 0.441 μg/ml, and the limits of quantitation were 0.055 and 1.336 μg/ml for MON and FEX, respectively. The developed method was applied successfully for the determination of the two drugs in their newly released fixed-dose combination prescribed for the treatment of allergic rhinitis. The mean per cent recoveries were found to be 100.680 ± 0.890 and 100.110 ± 0.940 for MON and FEX, respectively. Furthermore, the method was found to be eco-friendly green as was evaluated according to the Green Analytical Procedure Index tool guidelines and analytical eco-scale.     

Simultaneous determination of metolazone and losartan in their combined tablets using synchronous fluorescence spectroscopy

Synchronous spectrofluorimetry is utilized to carry out a rapid, sensitive and reliable method for determination of the binary mixture of metolazone (MTL) and losartan potassium (LSP). Under optimized experimental conditions, the synchronized fluorescence spectra of the two drugs were measured at Δλ = 80 nm in acidic methanolic solution and intensities were recorded at 260 nm for MTL and 335 nm for LSP. Linear correlation between fluorescence intensity and concentration were obtained through the ranges 0.02–0.2 μg/mL and 0.2–2.0 μg/mL for MTL and LSP, respectively. Limits of detection were 3.02 and 0.12 ng/mL, whereas limits of quantification were 9.16 and 0.35 ng/mL for MTL and LSP, respectively. The designated procedure was easily and successfully adopted to determine the two compounds in their single, as well as in co‐formulated, tablets and the results showed high precision and accuracy without any significant interference from common tablet excipients. A comparison of the obtained results with a published reference method was carried out and both showed good agreement with respect to accuracy and precision.     

Micelle‐enhanced spectrofluorimetric method for determination of lacidipine in tablet form; application to content uniformity testing

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of lacidipine (LCP) in tablets. The proposed method is based on the investigation of the fluorescence spectral behavior of LCP in both sodium dodecyl sulphate (SDS) and the tween‐80 micellar system. In aqueous solutions of acetate buffer (pH 4.5), the fluorescence intensities of LCP were greatly enhanced (ca. 2.4 and 4.3 folds) in the presence of either SDS or tween‐80, respectively. The fluorescence intensity was measured at 444 nm after excitation at 277 nm using either SDS or tween‐80 as a surfactant. The fluorescence–concentration plots were rectilinear over the ranges of 50.0–500.0 ng/ml and 5.0–200.0 ng/ml with lower detection limits of 5.11 and 2.06 ng/ml and lower quantification limits of 17 and 6.87 ng/ml using SDS and tween‐80, respectively. The method was successfully applied to the analysis of LCP in commercial tablets and the results were in good agreement with those obtained with the comparison method. Furthermore, content uniformity testing of pharmaceutical tablets was also conducted. Copyright © 2014 John Wiley & Sons, Ltd.     

Highly sensitive spectrofluorimetric method for rapid determination of orciprenaline in biological fluids and pharmaceuticals

Orciprenaline sulphate (ORP) is a direct‐acting sympathomimetic with mainly beta‐adrenoceptor stimulant activity. It is used as a bronchodilator in the management of reversible airway obstruction. For the first time, a rapid highly sensitive spectrofluorimetric method is described that is relied on measuring the fluorescence spectra of ORP at acidic pH and without addition of any chemical reagents. The relative fluorescence intensity was measured at 310 nm and after excitation at 224 nm. ORP native fluorescence was calibrated in both water and acetonitrile as diluting solvents. The method was designed to estimate the drug in miscellaneous matrices with high accuracy and precision. Linear ranges of calibration curves were 30.0–400.0 ng/ml and 10.0–240.0 ng/ml in water and acetonitrile, respectively. The detection limits were calculated and reached as low as 3.3 and 3.1 ng/ml, respectively, representing the ultra‐sensitivity of the proposed method. This result permitted application of this method for spiked human plasma and urine and was used as a preliminary investigation with good percentage recovery (89.4–106.8%). The application was further extended to analyse ORP in its pharmaceutical formulations. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.     

Resonance Rayleigh scattering methods for the determination of chitosan with Congo red as probe

Two methods were presented for the sensitive and selective determination of chitosan (CTS) with Congo red (CR) as probe based on resonance Rayleigh scattering (RRS) intensities in health products. In weakly acidic buffer solution, the binding of CTS to CR, could result in the enhancement of the RRS intensities. Moreover, after adding OP emulsifier (octyl‐phenyl polyoxyethylene ether) to the system, the RRS intensities showed more significantly enhancement. The maximum RRS signals for the CTS–CR system and the CTS–CR–OP system were located at 380 nm and 376 nm, respectively. Under optimum experimental conditions, the increased RRS intensities (ΔI) of these two systems were linear to CTS concentration in the range of 0.40–8.00 μg/ml and 0.05–1.00 μg/ml. Their limits of detection (LOD) were 44.81 ng/ml and 6.99 ng/ml, which indicated that the latter system was more sensitive than the former. In this work, the optimum conditions and the effects of some foreign substances on the determination were studied. In addition, the effect of the molecular weight of CTS and the reasons for the enhancement of resonance light scattering were discussed. Finally, these two methods were applied to the determination of chitosan in health products with satisfactory results.     

Simultaneous determination of piroxicam and norfloxacin in biological fluids by high‐performance liquid chromatography with fluorescence detection at zero‐order emission mode

A new highly sensitive high‐performance liquid chromatographic method with fluorescence detection (HPLC–FLD) in zero‐order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero‐order mode for emission. The zero‐order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001–20 μg/ml and 0.00003–0.035 μg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10^?4 and 1.32 × 10^?5 μg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10^?3 and 4.01 × 10^?5 μg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF.     

Spider diagram with greenness evaluation metrics for assessing the new synchronous spectrofluorimetric determination of bicalutamide and resveratrol in human plasma

A simple, selective, and eco-friendly synchronous fluorescence approach was introduced for the first time for the concurrent estimation of the anticancer combination therapy of bicalutamide and resveratrol. The method relies on measuring the synchronous fluorescence spectra of bicalutamide and resveratrol at 269 and 320 nm, respectively, using Δλ of 60 nm with ethanol as a green diluting solvent. The procedure was optimized, and the method was then fully validated. Excellent linearity (R² > 0.999) with very low detection limits (0.044 and 2.001 ng/ml) were obtained for both drugs, allowing for their analysis in human plasma. The green profile of the suggested approach was evaluated using the green solvents selecting tool (GSST), spider diagram for greenness index assessment, green analytical process index (GAPI), and Analytical GREEnness (AGREE) metric tools. These assessment metrics confirmed that the developed approach met the maximum number of green requirements, recommending its application as a green substitute for the regular analysis of the concerned drugs in human plasma. The simplicity of sample measurement enables and substantially accelerates the analysis, resulting in lower costs, enhanced procedure accuracy, and lower environmental effect.     

Combinational biosynthesis of dual-functional streptavidin-phycobiliproteins for high-throughput-compatible immunoassay

The development of fluorescent tools with desired fluorescence and efficient targeting is of great importance in the high-throughput immunoassay. Here we report combinational biosynthesis of new dual-functional streptavidin-phycobiliproteins (SA-PBPs) in Escherichia coli by fusing streptavidin with phycobiliproteins. These recombinant proteins can achieve a purity over 95% after one-step purification, and their maximum absorption and fluorescence emission wavelength are at 556 nm and 568 nm for SA-PCA-PEB (streptavidin-phycocyanin α subunit-phycoerythrin), and 624 nm and 646 nm for SA-PCA-PCB (streptavidin-phycocyanin α subunit-phycocyanobilin), respectively. The potential application of these dual-functional SA-PBPs in immunoassay was evaluated using “sandwich” ELISA method for detection of two biomarkers of liver cancers, i.e. α-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As a result, both SA-PCA-PCB and SA-PCA-PEB showed a good linear function with AFP & CEA within 0–50 ng/ml concentrations. Their limits of detection (LODs) for AFP and CEA were 0.25 ng/mL and 0.28 ng/ml using SA-PCA-PEB, and 1.01 ng/ml and 1.12 ng/ml using SA-PCA-PCB respectively. These results indicate that these novel dual-functional SA-PBPs are useful tools for a wide variety of immunoassay, and may have the advantages in their higher expandability and compatibility with existing and future immunoassay technologies.     

Enantiomeric assay of escitalopram S(+)‐enantiomer and its “in‐process impurities” using two different techniques

The enantiomeric purity of escitalopram oxalate ESC and its “in‐process impurities,” namely, ESC‐N‐oxide, ESC‐citadiol, and R(?)‐enantiomer were studied in drug substance and products using high‐performance liquid chromatography (HPLC)‐UV (Method I), synchronous fluorescence spectroscopy (SFS) (Method IIA), and first derivative SFS (Method IIB). Method I describes as an isocratic HPLC‐UV for the direct resolution and determination of enantiomeric purity of ESC and its “in‐process impurities.” The proposed method involved the use of α_l‐acid glycoprotein (AGP) chiral stationary phase. The regression plots revealed good linear relationships of concentration range of 0.25 to 100 and 0.25 to 10 μg mL^?1 for ESC and its impurities. The limits of detection and quantifications for ESC were 0.075 and 0.235 μg mL^?1, respectively. Method II involves the significant enhancement of the fluorescence intensities of ESC and its impurities through inclusion complexes formation with hydroxyl propyl‐β‐cyclodextrin as a chiral selector in Micliavain buffer. Method IIA describes SFS technique for assay of ESC at 225 nm in presence of its impurities: R(?)‐enantiomer, citadiol, and N‐oxide at ?λ of 100 nm. This method was extended to (Method IIB) to apply first derivative SFS for the simultaneous determination of ESC at 236 nm and its impurities: the R(?)‐enantiomer, citadiol, and N‐oxide at 308, 275, and 280 nm, respectively. Linearity ranges were found to be 0.01 to 1.0 μg mL^?1 for ESC and its impurities with lower detection and quantification limits of 0.033/0.011 and 0.038/0.013 μg mL^?1 for SFS and first derivative synchronous fluorescence spectra (FDSFS), respectively. The methods were used to investigate the enantiomeric purity of escitalopram.     

Fast concurrent determination of guaifenesin and pholcodine in formulations and spiked plasma using first derivative synchronous spectrofluorimetric approach

Guaifenesin and pholcodine are frequently co-formulated in certain dosage forms. A new fast first derivative synchronous spectrofluorometric method has been used for their simultaneous analysis in mixtures. Here, first derivative synchronous spectrofluorometry enabled the successful simultaneous estimation of guaifenesin at 283 nm and pholcodine at 275 nm using a wavelength difference (Δλ) of 40 nm. The method was fully validated following International Council of Harmonization guidelines. For guaifenesin and pholcodine, linearity was determined within the corresponding ranges of 0.05–0.30 and 0.10–6.0 μg/ml. The two drugs were effectively analyzed using the developed approach in their respective formulations, and the results showed good agreement with those attained using reference methods. The method demonstrated excellent sensitivity, with detection limits down to 0.007 and 0.030 μg/ml and quantitation limits of 0.020 and 0.010 μg/ml for guaifenesin and pholcodine, respectively. Therefore, the procedure was successful in determining these drugs simultaneously in vitro in spiked plasma samples and syrup dosage form. The developed methodology also offered an environmentally friendly advantage by utilizing water as the optimal diluting solvent throughout the whole work. Different greenness approaches were investigated to ensure the method’s ecofriendly properties.     

Simultaneous determination of desloratadine and montelukast sodium using second‐derivative synchronous fluorescence spectrometry enhanced by an organized medium with applications to tablets and human plasma

A rapid, simple, and sensitive second‐derivative synchronous fluorimetric method has been developed and validated for the simultaneous analysis of a binary mixture of desloratadine (DSL) and montelukast sodium (MKT) in their co‐formulated tablets. The method is based on measurement of the synchronous fluorescence intensities of the two drugs in McIlvaine's buffer, pH 2.3, in the presence of carboxy methyl cellulose sodium (CMC) as a fluorescence enhancer at a constant wavelength difference (Δλ) of 160 nm. The presence of CMC enhanced the synchronous fluorescence intensity of DSL by 216% and that of MKT by 28%. A linear dependence of the concentration on the amplitude of the second derivative synchronous fluorescence spectra was achieved over the ranges of 0.10–2.00 and 0.20–2.00 µg/mL with limits of detection of 0.02 and 0.03, and limits of quantification of 0.05 and 0.10 µg/mL for DSL and MKT, respectively. The proposed method was successfully applied for the determination of the studied compounds in laboratory‐prepared mixtures and tablets. The results were in good agreement with those obtained with the comparison method. The high sensitivity attained by the proposed method allowed the determination of MKT in spiked human plasma with average % recovery of 100.11 ± 2.44 (n = 3). Copyright © 2014 John Wiley & Sons, Ltd.     

First‐derivative synchronous spectrofluorimetric method for estimation of losartan potassium and atorvastatin in their pure forms and in tablets

Losartan potassium (LOS) and atorvastatin (ATR) are used in combination for long‐term treatment of stroke and for treatment of hypertension with high‐level cholesterol. Both drugs were simultaneously determined and validated using a novel, easy, fast, and economical first‐derivative synchronous fluorescence spectroscopic method. Methanol was used as the solvent for both drugs at a Δλ 80 nm and with a scanning rate of 600 nm/min. Peaks were determined as at 288.1 nm and 263.6 nm for LOS and ATR, respectively. The proposed method was validated according to International Conference on Harmonization guidelines and, subsequently, the developed method was applicable to the analysis of the two compounds in their different formulations without interference from each other. Amplitude–concentration plots were rectilinear over the concentration ranges 1.0–10.0 μg/ml and 0.5–5.0 μg/ml for LOS and ATR, respectively. Detection limits were found to be 0.096 μg/ml and 0.030 μg/ml and quantitation limits were 0.291 μg/ml and 0.093 μg/ml for LOS and ATR, respectively. The proposed method was successfully applied to the analysis of both compounds in synthetic mixtures and in laboratory‐prepared tablets. These results were in accordance with the results acquired using the comparison method, high‐performance liquid chromatography.     

Second‐derivative synchronous spectrofluorimetric determination of nebivolol hydrochloride and amlodipine besylate in their combined dosage form

A rapid, simple, accurate and highly sensitive spectrofluorimetric method was developed for the simultaneous analysis of nebivolol hydrochloride (NEB) and amlodipine besylate (AML). The method was based on measuring the synchronous fluorescence intensity of the drugs at Δλ = 40 nm in methanol. Various experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully studied and optimized. The calibration plots were rectilinear over concentration ranges of 0.05–1.5 µg/mL and 0.5–10 µg/mL for NEB and AML with limits of detection (LOD) of 0.010 and 0.051 µg/mL and limits of quantitation (LOQ) of 0.031 and 0.156, respectively. The peak amplitudes (²D) of the second derivative synchronous fluorimetry (SDSF) were estimated at 282 nm for NEB and at 393 nm for AML. Good linearity was obtained over the concentration ranges. The proposed method was successfully applied to the determination of the studied compounds in laboratory‐prepared mixtures, commercial single and laboratory‐prepared tablets. The results were in good agreement with those obtained using the comparison method. The mean percent recoveries were found to be 100.12 ± 0.77 and 99.91 ± 0.77 for NEB and AML, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Spectrofluorimetric determination of amisulpride and bumidazone in raw materials and tablets

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of amisulpride (AMS) and bumidazone (BUM) in tablet form. The proposed method is based on measuring the native fluorescence of the studied drugs in methanol at 360 and 344 nm after excitation at 276 and 232 nm for AMS and BUM, respectively. The fluorescence–concentration plots were rectilinear over the ranges of 5.0–60.0 ng/mL for AMS and 0.5–5.0 µg/mL for BUM. The lower detection limits were 0.70 ng/mL and 0.06 µg/mL, and the lower quantification limits were 2.0 ng/mL and 0.18 µg/mL for AMS and BUM, respectively. The method was successfully applied for the analysis of AMS and BUM in commercial tablets. Statistical evaluation and comparison of the data obtained using the proposed and comparison methods revealed good accuracy and precision for the proposed method. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of busulfan in human plasma using high-performance liquid chromatography with pre-column derivatization and fluorescence detection

A rapid, sensitive and reproducible high-performance liquid chromatographic assay for busulfan in human plasma was developed. After extraction of plasma samples with acetonitrile and methylene chloride, busulfan and the internal standard [1,5-bis(methanesulfonyloxy)pentane] were derivatized with 8-mercaptoquinoline to yield fluorescent compounds which were detected with a fluorescence detector equipped with filters of 360 nm (excitation) and 425 nm (emission). Calibration graphs showed a linear correlation (r>0.9990) over the concentration range of 20–2000 ng/ml. The recovery of busulfan from plasma standards was 70±5%. The detection and quantification limits for busulfan in plasma samples were established at 9 ng/ml and 20 ng/ml, respectively. The intra- and inter-assay variations were lower than 8% and 10%, respectively. The applicability of the method was verified by analyzing the plasma concentrations of busulfan in a patient to whom it was administered orally on two different days.     

Quantification of three triterpenic acids in dried rosemary using HPLC‐fluorescence detection and 4‐(4,5‐diphenyl‐1H‐imidazole‐2‐yl)benzoyl chloride derivatization

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4‐(4,5‐diphenyl‐1H‐imidazole‐2‐yl)benzoyl chloride (DIB‐Cl) and detected using HPLC‐fluorescence (FL). Dried rosemary (50 mg) was ground, added to 3 ml of ethanol, sonicated for 40 min, then the sample solution was added to a mixture of 1% trimethylamine and 1 mM DIB‐Cl in acetonitrile. The mixture was settled for 5 min at room temperature, then the DIB‐triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250 × 4.6 mm, 6 μm) eluted with 25 mM acetate buffer (pH 4.5)/methanol/acetonitrile (= 8:10:82 v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λ_ex) and 490 nm (λ_em) and the maximum retention time of the derivatives was 30 min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r ≥ 0.997) in the range 2.5–100 ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5 ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.