Hanan Merey

A novel, sensitive, and green micellar UPLC method was proposed and validated for the simultaneous
determination of four hypoglycemic agents used in type II diabetes mellitus treatment namely, pioglitazone,
alogliptin, glimepiride, and vildagliptin. The developed UPLC method was successfully applied for quantitative
analysis of these drugs in bulk, in pharmaceutical formulations, and in spiked human plasma. Chromatographic
separation was carried out on a Kinetex® 1.7 μm XB-C18 100 Å (50 × 2.1 mm) column, using a degassed and filtered
mixture of (0.1 M SDS- 0.3% triethyl amine- 0.1% phosphoric acid (pH 6)) and n-propanol (85:15 v/v), at a flow rate
of 0.2 mL/min. The experimental conditions of the suggested method were well investigated and optimized. The
newly developed micellar UPLC method is capable of determining different dosage forms at the same time with
the same solvents, saving time and effort. The method was found to be efficiently applicable in spiked human
plasma and could be extended to study the pharmacokinetics of the cited drugs in real human plasma samples.
The greenness of the developed method was evaluated by applying the Eco-scale scoring tool, which verified the
excellent greenness of the analytical method.

Green electro-analytical method was developed and validated for quantitative determination and monitoring of the dissolution behavior of Losartan potassium tablets by in-line potentiometric measurement system with no need for pre-treatment or derivatization of the sample. A sensor was fabricated for in-line determination of Losartan potassium LOS in its dissolution medium utilizing polyvinyl chloride (PVC) based membrane and nitrophenyl octyl ether (NPOE) as a plasticizer. Tetraheptyl ammonium bromide (THAB) was employed as anion exchanger for the first sensor, while cetyltrimethylammonium bromide (CTAB) was used in the second sensor. The proposed method showed fast, stable near Nernstian responses across a relatively wide LOS concentration range (1.0 × 10-5 to 1.0 × 10-2 mol/L) in the case of THAB-based sensor while LOS concentration range was (1.0 x 10-4 - 1.0 x 10-2 mol/L) in case of CTAB-based sensor. The dissolution method
was developed according to FDA regulations using USP apparatus II, 50 rpm rotation speed, at 37.0 ± 0.5°C and 900 mL of deareated double distilled water as the dissolution medium. Dissolution profile was generated over 45 min and compared to those obtained by the official spectrophotometric and HPLC methods. The developed method can be efficiently applied as benchtop real-time in-process analysis of LOS concentration. Greenness assessment using Analytical Eco-scale, National Environmental Method Index (NEMI), Green Analytical Procedure Index (GAPI) and Analytical Greenness (AGREE) calculator was conducted to evaluate and compare the proposed method with the official ones.

BACKGROUND: Paracetamol (PC) is one of the most widely used analgesic and antipyretic drugs and has recently been integrated into the supportive therapy of COVID-19. Pharmaceuticals containing methionine (MT) with PC may contribute to avoid hepatotoxicity and eventual PC overdose-dependent death.

OBJECTIVE: The current work purposes to develop and validate two chromatographic methods for the simultaneous determination of MT and PC in the presence of two PC impurities (4-nitrophenol [NP] and 4-aminophenol [AP]).

METHOD: Two chromatographic methods were established and validated according to the International Conference on Harmonization guidelines. The first one was an RP-HPLC/UV method based on applying a "dual-mode" gradient elution. The separation was realized via varying both the composition of the ternary mobile phase (acetonitrile-methanol-water) and its flow rate. This strategy enabled a relatively rapid analysis with a satisfactory resolution, although the investigated compounds exhibit a significant difference in lipophilicity. The second one relied on TLC-densitometry, where the optimum separation was realized using a quaternary mobile phase system composed of butanol-dioxane-toluene-methanol (8:2.5:3.5:0.3, by volume). Both methods were monitored at 220 nm.

RESULTS: The developed methods were proven to be robust, accurate, specific, and appropriate for the routine analysis of PC in its pure form or in pharmaceutical formulations with MT in quality control laboratories.

CONCLUSIONS: The corresponding methods are suitable to determine MT and PC in the presence of PC impurities.

HIGHLIGHTS: The study achieves the analysis of MT and PC in the presence of PC impurities via the application of HPLC and TLC-densitometry methods.

Empagliflozin and linagliptin are newly approved FDA combination that used for the treatment of type 2 diabetes mellitus (T2DM) under trade name Glxambi. Two spectroflourimetric methods were developed for simple quantitative determination of empagliflozin and linagliptin in their pharmaceutical formulation and human plasma without need any tedious processing operations. Empagliflozin has a native fluorescence nature, therefore can be directly determined by measuring emission peak at 305 nm after excitation at 234 nm. There is no any interference from linagliptin at this emission wavelength. On the other hand, linagliptin is a very weak florescent compound that needs to react with fluorogenic reagent to be quantitatively determined without any reaction of empagliflozin. So, quantitative analysis of linagliptin was achieved by coupling with NBD-Cl which is an electro active halide reagent (targeting only Linagliptin with no effect on empagliflozin). Dark yellow fluorophore with high fluorescence is a result of this reaction and can be measured at emission wavelength 538 nm after excition at wavelength 469 nm. Experimental conditions of the suggested methods were well checked and optimized. The regression plots were found to be linear over the range of 40-1200 ng/mL and 3-700 ng/mL for empagliflozin and linagliptin, respectively. The obtained results by the suggested methods were statistically compared with those obtained by the reported methods, showing no significant difference with respect to accuracy and precision at p = 0.05.

A new and simple spectrophotometric method was developed for the simultaneous determination of a new antidiabetic mixture of linagliptin and empagliflozin namely fourier self deconvulated method. The developed method based on minimal mathematical data processing on the zero order spectrum for solving sever overlapping spectra of the mentioned drugs in their pure forms and pharmaceutical dosage form. The zero order spectra of linagliptin and empagliflozin were deconvulated using Fourier transforms function. The peak amplitudes at 232 nm were selected for linagliptin and at 239 nm for empagliflozin. The constructed calibration graphs were linear over the range (5-30 µg/mL) and (2-12 µg/mL) for empagliflozin and linagliptin, respectively. The adopted method was simple, accurate, precise and validated according to the ICH guidelines.

Two chromatographic methods were validated for the determination of the widely prescribed analgesic and antipyretic drug combination of paracetamol (PC) (recently integrated into the supportive treatment of COVID-19), propyphenazone (PZ) and caffeine (CF) in the presence of two PC impurities, namely 4-aminophenol and 4-nitrophenol. A "dual-mode" gradient high-performance liquid chromatography method was developed, where the separation was achieved via "dual-mode" gradient by changing both the ternary mobile phase composition (acetonitrile: methanol: water) and the flow rate. This enables a good resolution within a relatively shorter analysis time. The analysis was realized using Zorbax Eclipse XDB column C18, 5 μm (250 × 4.6 mm) and the UV detector was set at 220 nm. The other method is a thin-layer chromatography densitometry method, where the separation was achieved using a mobile phase composed of chloroform: toluene: ethyl acetate: methanol: acetic acid (6: 6: 1: 2: 0.1, by volume). Densitometric detection was performed at 220 nm on silica gel 60 F254 plates. The developed methods were fully validated as per the ICH guidelines and proved to be accurate, robust, specific and suitable for application as purity indicating methods for routine analysis of PC in pure form or in pharmaceuticals with PZ and CF in quality control laboratories.

Kinetics studies are important in quality control to assure the safety and efficacy of the pharmaceutical compounds.
Recently, potentiometric ion-selective electrodes (ISEs) breakthrough kinetics study due to their ability
to provide real-time measurements which meet “green analytical chemistry” (GAC) principles. A polyvinyl
chloride (PVC) matrix membrane was fabricated to monitor bromhexine (BR) oxidative degradation kinetics
using tetraphenylborate as a cation exchanger. At-line monitoring of the oxidative degradation kinetics of BR was
performed; by continuous measuring the decrease in the sensor potential over time. So a real-time observation
and a continuous profile were obtained for the oxidation of BR under various H2O2 concentrations and temperature.
This kinetic study determines the oxidation activation energy that was 6.4 kcal mol−1. To expand the
application of this sensor, BR was determined in bulk powder and dosage form in the presence of both coformulated
drug (acefylline piperazine) and dosage form additives. The proposed sensor had been characterized
according to IUPAC recommendations and a linear dynamic range was 5×10−6 to 1×10−3M and other sensor
parameters had been calculated.

Three green, simple, precise, accurate and sensitive spectrophotometric methods were developed for the determination
of a binary mixture of lidocaine hydrochloride (LDC) and cetylpyridinium chloride (CPC) in the presence
of dimethylaniline (DMA). In the three methods, the interference of DMA spectrum is eliminated using
the ratio subtraction method. Method (A) depended on determining LDC and CPC by measuring the first derivative
of the ratio spectra (1DD) at 271.0 and 268.4 nm, respectively. Method (B) was the ratio difference (RD),
based on dividing the absorption spectrum of the binary mixture by a standard spectrum of CPC or LDC, then
measuring the amplitude difference of the ratio spectra (ΔP) between 231.2 and 240.0 nm for LDC and between
242.8 and 258.0 nm for CPC. Method (C) based on the application of dual wavelength coupled with the
isoabsorptive point method. This was achieved by measuring the absorbance difference (ΔA) between 243.0
and 268.6 nm for the determination of LDC, followed by application of isoabsorptive point method comprised
ofmeasurement the total content of the mixture of LDC and CPC at their isoabsorptive point at 240.0nm. The content
of CPCwas obtained by subtraction. The specificity of the developed methods was investigated by analyzing
laboratory prepared mixtures containing different ratios of LDC and CPC in presence of DMA. The proposed
methods displayed useful analytical characteristics for the determination of LDC and CPC in bulk powder and
their combined dosage form. The obtained results were statistically comparedwith those obtained by the official
methods, showing no significant difference with respect to accuracy and precision.

Two chromatographic methods were developed, optimized and validated for simultaneous determination of calcipotriol monohydrate (CPM) and betamethasone dipropionate (BMD) in the presence of two dosage form additives named; butylated hydroxytoluene (BHT) and alpha-tocopherol (TOCO). The proposed methods were accurate, sensitive and specific. The first method based on using aluminum thin-layer chromatographic plates precoated with silica gel GF254 as a stationary phase and chloroform-ethyl acetate-toluene (5:5:3, by volume) as a developing system. This was followed by densitometric measurement of the separated bands at 264 nm. Whereas the second method is RP-HPLC where OnyxMonolithic C18® column was used with a gradient profile using methanol, water and acetic acid at flow rate 2.0 mL min-1. Detection was carried out at 264 nm. The methods were validated according to ICH guidelines. The specificity of the developed methods was investigated by analyzing the pharmaceutical dosage form. The validity of the proposed methods was assessed using the standard addition technique. The obtained results were statistically compared with those obtained by the official methods, showing no significant difference with respect to accuracy and precision at P = 0.05.

A simple, rapid and sensitive ultra-performance liquid chromatography/positive ion electrospray tandem mass

spectrometric method was developed and validated for the quantification of captopril in human plasma.

Following plasma protein precipitation, the analyte and internal standard Rosuvastatin were separated by ultra-
performance liquid chromatography using a gradient mode mobile phase on a reversed-phase column and

analyzed by mass spectrometry in the multiple reaction monitoring mode (MRM) using the respective [MRH]R

ions, m/z 218.09/116.16 for captopril and m/z 482.2/ 258.17 for the internal standard. The method exhibited a

linear dynamic range of 10–2000 ng/mL for captopril in human plasma. The lower limit of quantification

was 10 ng/mL with a relative standard deviation of less than 6%. Acceptable precision and accuracy were

obtained for concentrations over the standard curve range. A total run time of 3 minutes for each sample made it

possible to analyze more than 20 human plasma samples per one hour. The validated method has been

successfully applied to analyze human plasma samples in a bioequivalence study of captopril after oral

administration of 50 mg captopril tablet to 24 healthy subjects.

Antipyrine and benzocaine are formulated together for the treatment of ear inflammation
and to relieve pain. Four spectrophotometric methods were developed for the simultaneous
determination of antipyrine (AN) and benzocaine (BE) in their combined dosage form. Method
A depends on applying dual wavelength method where antipyrine was determined by measuring
the absorbance at 254.1 and 309.1 nm (corresponding to zero difference of benzocaine), while the
absorbance difference at 230.1 and 263.5 nm (corresponding to zero difference of antipyrine) was
selected for benzocaine determination in the laboratory prepared spectrum. Method B depends
on measuring the peak amplitude of first derivative at 305 nm for calculating benzocaine concentration
then the total concentration of both drugs was determined using isoabsorptive point at
257.4 nm (antipyrine concentration was then calculated by subtraction). Method C is based on measuring
the peak difference of the ratio spectra at Dp (239.1–285 nm) and Dp (301.4–250 nm) for the
determination of antipyrine and benzocaine, respectively. Method D depends on measuring peak to
peak amplitude of the first derivative of ratio spectra at (234.5 + 244.2 nm) and peak amplitude at
295.5 nm for the determination of antipyrine and benzocaine, respectively. The proposed methods
were validated and applied for the analysis of antipyrine and benzocaine in their laboratory prepared
mixtures and pharmaceutical formulation. Statistical comparison between the results of
the proposed methods and those of the reported methods showed no significant difference.

Fluticasone propionate (FLU) and Azelastine hydrochloride (AZE) are co-formulated with phenylethyl alcohol
(PEA) and Benzalkonium chloride (BENZ) (as preservatives) in pharmaceutical dosage form for treatment of seasonal
allergies. Different spectrophotometric methods were used for the simultaneous determination of cited
drugs in the dosage form. Direct spectrophotometric method was used for determining of AZE, while Derivative
of double divisor of ratio spectra (DD-RS), Ratio subtraction coupled with ratio difference method (RS-RD) and
Mean centering of the ratio spectra (MCR) are used for the determination of FLU. The linearity of the proposed
methods was investigated in the range of 5.00–40.00 and 5.00–80.00 μg/mL for FLU and AZE, respectively. The
specificity of the developed methods was investigated by analyzing laboratory prepared mixtures containing different
ratios of cited drugs in addition to PEA and their pharmaceutical dosage form. The validity of the proposed
methods was assessed using the standard addition technique. The obtained results were statistically compared
with those obtained by official or the reported method for FLU or AZE, respectively showing no significant difference
with respect to accuracy and precision at p = 0.05.

Mometasone Furoate (MO) and Miconazole Nitrate (MI) are co-formulated with p-chlorometa-cresol
(CMC) which added in pharmaceutical dosage form as a preservative together for treatment of skin
infections by candida or dermatophyte in which inflammatory and pruritic manifestation are prominent.
Comparative study between different specrtophotometric methods were done for the simultaneous determination
of cited drugs in the dosage form. Spectrophotometric methods were used including, derivative ratio spectra
(DD), derivative of double divisor of ratio spectra (DD-RS) and Mean centering of the ratio spectra (MCR).
While the chemometric methods were used including, partial least-squares (PLS) and principal component
regression (PCR). The linearity of the proposed methods was investigated in the range of 3-33 and 60-840 μg/
mL for MO and MI, respectively. The specificity of the developed methods was investigated by analyzing
laboratory prepared mixtures containing different ratios of cited drugs in addition to CMC and their pharmaceutical
dosage form. The validity of the proposed methods was assessed using the standard addition technique. The
obtained results were statistically compared with those obtained by the reported method, showing no significant
difference with respect to accuracy and precision at p = 0.05.

Two chromatographic methods were developed and validated for the simultaneous
determination of Mometasone furoate (MO) and Formoterol fumarate dihydrate (FOR). Combination
of MO and FOR is used for the treatment of asthma in patients suffering from reversible
obstructive airway disease. The first chromatographic method was based on using aluminum
TLC plates pre-coated with silica gel GF254 as the stationary phase and chloroform:ethyl acetate:
methanol:toluene:formic acid (5:2:2:2:0.1, by volume) as the mobile phase followed by densitometric
measurement of the separated bands at 233 nm. The second method is a high performance liquid
chromatographic method for the separation and determination of MO and FOR using reversed
phase C18 column with isocratic elution. The mobile phase composed of methanol: 0.5% ammonium
acetate pH adjusted with acetic acid (80:20, v/v) at a flow rate of 1.0 mL/min. Quantitation
was achieved with UV detection at 220 nm. The specificity of the developed methods was investigated
by analyzing the pharmaceutical dosage form. The validity of the proposed methods was
assessed using the standard addition technique. The obtained results were statistically compared
with those obtained by the reported methods, showing no significant difference with respect to accuracy
and precision at p = 0.05.