# Fluorometric Determination of Drugs Containingα-Methylene Sulfoxide Functional Groups Using N1- Methylnicotinamide Chloride as a Fluorogenic Agent

Authors: Khaled M. Elokely; Mohamed A. Eldawy; Mohamed A. Elkersh; Tarek F. El-Moselhy
Journal: ISRN Analytical Chemistry
 (2012)

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## Abstract

A simple fluorometric method, usingN1-methylnicotinamide chloride (NMNCl) as a fluorogenic reagent, has been developed, adapted, and validated for the quantitative estimation of drugs containing α-methylene sulfoxide functional groups. The proposed method has been applied successfully to the determination of sulindac (1), omeprazole (2), lansoprazole (3), pantoprazole (4), and rabeprazole (5) in the pure form, laboratory-prepared mixtures, pharmaceutical dosage forms, spiked human plasma samples, and in hospitalized patient's or volunteer's blood.
For the standard solutions of 1, 2, 3, 4, and 5, the method showed linearity over concentration ranging between 1–50 μg/mL, 50–1200 ng/mL, 100–1500 ng/mL, 10–1500 ng/mL, and 20–2200 ng/mL, respectively. For the spiked human plasma of 1, 2, 3, 4, and 5, the linearity was shown over concentration ranging between 1–50 μg/mL, 75–1200 ng/mL, 100–1400 ng/mL, 10–1500 ng/mL, and 50–2100 ng/mL, respectively. The method showed good accuracy, specificity, and precision in both laboratory-prepared mixtures and spiked human plasma samples.
The proposed method is simple, does not need sophisticated instrumentation, suitable for quality control application, bioavailability, and bioequivalency studies. Besides, the sensitivity and detection limits are comparable to sophisticated chromatographic methods.


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## Body

## 1. Introduction

Nakamura described the application of a fluorometric method of the reaction ofN1-methylnicotinamide chloride (NMNCl) for the analysis of various compounds containing α-methylene carbonyl functional groups [1]. Nakamura also made qualitative tests on the reaction mechanism and identified the cyclized α-adduct fluorophore [1]. This reaction was not tested before for compounds containing α-methylene adjacent to other functional groups.Encouraged by the successful application of the NMNCl methodology to the determination of similar toα-methylene carbonyl functional group-containing drugs, namely, warfarin [2], pentoxifylline, propafenone hydrochloride, and acebutolol hydrochloride [3] and the almost isosteric α-methylene sulfone/sulfonamide group, such as methyl sulfonyl methane (MSM), tinidazole, and rofecoxib [4], we began to adapt, validate, and extend the applicability of this methodology for the analysis of sterically hindered cyclic ketones such as ketamine hydrochloride, griseofulvin, and levonorgestrel (unpublished results).In this paper, we report the application of this methodology for determination ofα-methylene sulfoxide functional group-containing drugs such as the nonsteroidal anti-inflammatory drug (NSAID) sulindac (1) and the proton pump inhibitors (PPIs) omeprazole (2), lansoprazole (3), pantoprazole (4), and rabeprazole (5).Compound1 is a nonsteroidal anti-inflammatory indene derivative [5]. Compounds 2–5 are proton pump inhibitors (PPIs) [6].Several methods have been reported for the analysis of1 that may include UV spectrophotometry [7], reversed-phase HPLC as a stability-indicating assay [8], other HPLC methods [9, 10], enzyme immunoassay [11], and capillary electrophoresis [12, 13].Several methods have been developed for determination, chiral separation, stability, or pharmacokinetic studies of2 in bulk form, pharmaceuticals or biological samples, and these methods may include different spectrophotometric methods via formation of metal chelates [14], UV-derivative spectrophotometry [15] and HPLC methods for its determination [16], separation of enantiomers [17], for stability studies [18], or for the study of its pharmacokinetic profile [19].Several methods have been reported for the analysis of3 that may include UV/visible spectrophotometry [20] and HPLC for its determination [21], enantiomeric separation [22], or pharmacokinetic study [23].Several methods have been reported for the analysis of4 that may include kinetic spectrophotometry using 1-fluoro-2,4-dinitrobenzene [24], UV spectrophotometry [20], and different HPLC methods for its determination [25] or for its metabolites [26].Several methods have been reported for the analysis of5 that may include HPLC [27] and HPLC with NMR detection [28].Recently published methods described the simultaneous estimation of2–5 are also reported [29, 30].The objective of this work is to develop, adapt, and validate a simple fluorometric method that can be applied in the determination ofα-methylene sulfoxide group-containing drugs, namely, drugs1–5 in pure form, dosage forms, spiked human plasma samples and in patient’s blood. The proposed method has minimal instrumentation and chemical requirements; nevertheless, its sensitivity and specificity are comparable to other elaborated chromatographic techniques. In addition, it is a versatile method that can find applications on a wide range of pharmaceutical preparations and biological fluids.


## 2. Experimental

### 2.1. Apparatus

Shimadzu RF 5301 PC spectrofluorometer.


### 2.2. Materials

#### 2.2.1. Authentic Drugs

Working standards of1,2,3,4, and 5 were supplied by Sigma Pharmaceutical Industries, Egypt, Amyria Pharmaceutical Industries, Alexandria, Egypt, T3A Pharma, Egypt, Medical Union Pharmaceuticals (MUP), Egypt, and Global Napi Pharmaceuticals (GNP), Egypt, respectively.Plasma samples were purchased from the Central Blood Bank of Tanta University Hospital.


#### 2.2.2. Other Chemicals

N1-Methylnicotinamide chloride was purchased from Sigma Chemicals Co. Formic acid, sodium hydroxide, methanol, and all other chemicals were of analytical grade. Water used was doubly distilled.


### 2.3. Dosage Forms

#### 2.3.1. Sulindac(1)

Rudac tablet (Sigma Pharmaceutical Industries) was labeled to contain 150 and 200 mg, HiDac tablet (Hipharm for Manufactured Pharmaceuticals) labeled to contain 200 mg.


#### 2.3.2. Omeprazole(2)

Gastrazole capsules, 20 mg (Amyria Pharmaceutical Industries), Epirazole capsules, 20 mg (Egyptian International Pharmaceutical Industries Co. (EIPICO)), Omepak capsules, 10 and 20 mg (Sedico Pharmaceutical Co.), Pepzole capsules, 40 mg (Alkan Pharma), Gasec capsules, 20 mg (Mepha), Omez capsules, 10 mg (Pharaonia Pharmaceuticals PharoPharma), Gastrocure capsules, 20 mg (October Pharma), Napizole capsules, 20 mg (Global Napi Pharmaceuticals (GNP)), Ulstop capsules, 20 mg (Pharco Pharmaceuticals), Risek capsules, 20 mg (Julphar), Gastroloc capsules, 40 mg (Sigma), Omepral capsules, 20 mg (Memphis Co. for Pharmaceuticals and Chemical Industry), Trio capsules, 20 mg (Alkan Pharma), and Nexium tablets, 20 mg esomeprazole (Astra Pharmaceuticals).


#### 2.3.3. Lansoprazole(3)

Lansoprazole capsules, 15 mg (Rexcel), Zollipak capsules, 30 mg (Sedico), Peptazole capsules, 30 mg (Saudi Pharmaceutical Industries and Medical Appliances Corporation (Spimaco), Lanzor capsules, 15 and 30 mg (Aventis Pharma), and Lopral capsules, 30 mg (T3A Pharma).


#### 2.3.4. Pantoprazole(4)

Controloc tablets, 40 mg (Byk Gulden Lomberg Chemische Fabrik GmbH), Pantoloc tablets, 20 and 40 mg (Medical Union Pharmaceuticals (MUP)), and Pantazole tablets, 40 mg (Sigma Pharmaceutical Industries).


#### 2.3.5. Rabeprazole(5)

Bepra tablets, 20 mg (GNP) and Pariet tablets, 20 mg (Janssen Cilag).All these preparations were purchased from the local market of Egypt except for Nexium that was purchased from Saudi Arabia.


### 2.4. Reagents and Standard Solutions

#### 2.4.1. Stock Standard Solutions of Drugs

Stock standard solutions were prepared in 0.1 N sodium hydroxide for1, 2, and 4; 50% aqueous methanol for 3 and methanol for 5 to contain 100 mg/mL for 1, 150 μg/mL for 3, 4, and 5, and 250 μg/mL for 5.


#### 2.4.2. Serial Standard Solutions of Drugs

Aliquots of the stock solutions were diluted quantitatively with the same solvent to obtain concentration ranging between 10–500μg/mL for 1, 0.1–15 μg/mL for 2, 3, and 4, and 0.1–25 μg/mL for 5.


#### 2.4.3. Assay Solutions of Drugs in Synthetic Mixtures

Several synthetic mixtures were prepared for1, 4, and 5 to contain different proportions of the possible interfering substances that may be present with the drug in its dosage form. Three synthetic mixtures containing 1 were prepared. The first mixture contained 200 mg 1, 230 mg cellulose, 10 mg magnesium stearate, and 60 mg starch. The second mixture contained 200 mg 1, 100 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 130 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing4along with various additives and inactive ingredients were prepared. The first mixture contained 20 mg 4, 280 mg avicel, 60 mg maize starch, 10 mg magnesium stearate, 10 mg magnesium carbonate, 1 mg quinolone, 1 mg acidisol, 60 mg talc, 1 mg colloidal silicon, 6 mg polyethylene glycol 6000, 50 mg Eudragit L100-55, and 1 mg titanium dioxide. The second mixture contained 20 mg 4, 280 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 340 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing5 were prepared. The first mixture contained 20 mg 5, 300 mg croscarmellose sodium, 72 mg talc, 1 mg sodium hydroxide, 1 mg titanium dioxide, and 8 mg magnesium stearate. The second mixture contained 20 mg 5, 180 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 240 mg avicel instead of lactose and gelatin.Each synthetic mixture of1 and 4 was extracted with 100 mL 0.1 N sodium hydroxide. The synthetic mixture of 5 was extracted with 100 mL methanol. The extracts were filtered, and the first 10 mL of the filtrate were rejected. Aliquots of the filtrate were diluted with the same solvent to obtain serial solutions in the concentration ranges 10–500 μg/mL, 0.1–15 μg/mL, and 0.1–25 μg/mL for 1, 4, and 5, respectively.


#### 2.4.4. Assay Solutions of Drugs in Their Pharmaceutical Preparations

A quantity of the mixed contents of 20 capsules or tablets equivalent to one capsule or tablet of1–5 were finely powdered and transferred with the aid of several portions of 0.1 N sodium hydroxide solution (for 1, 2, and 4), 50% aqueous methanol (for 3), or methanol (for 5) to a 100 mL volumetric flask, and the volume was completed with the same solvent. The resulting solution was filtered and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain 200 μg/mL, 6 μg/mL, 3 μg/mL, 4 μg/mL, and 6 μg/mL solutions of 1–5, respectively.


#### 2.4.5. Assay Solutions of Drugs in Spiked Human Plasma Samples

Serial Standard Solutions of the Drugs
Serial standard solutions for1,2,3, and 4 were prepared in 0.1 N sodium hydroxide solution in concentrations ranging between 1–50 mg/mL for 1and 50–1500 μg/mL for 2,3, and 4. Serial standard solutions of 5 were prepared in methanol in concentrations ranges of 1–2500 μg/mL.Preparation of Spiked Human Plasma Samples
Two hundredμL of each 1,2,3, and 4 serial standard solution were diluted with 1800 μL human plasma and vortex mixed to obtain concentrations ranging between 0.1–5 mg/mL for 1 and 5–150 μg/mL for 2,3, and 4. For 5 200 μL of each serial standard solution were evaporated, the residue was dissolved in 1800 μL human plasma and vortex mixed, and 200 μL distilled water were added and vortex mixed to obtain 1–250 μg/mL.Preparation of Assay Solutions of Drugs in Plasma Samples
Two hundredμL of each spiked human plasma samples were mixed with 1800 μL methanol and centrifuged for 15 minutes to separate the precipitated protein. The clear supernatant was filtered through Millipore filter (0.45 μm) to obtain solutions in concentration ranging between 10–500 μg/mL for 1, 0.5–15 μg/mL for 2, 3, and 4, and 0.1–25 μg/mL for 5.Determination of1–5 in Volunteer’s Blood or Hospitalized Patient’s Blood
A blood sample was withdrawn in a test tube to which heparin was previously added and dried. The sample was centrifuged to separate plasma and then treated as previously mentioned under preparation of assay solutions of each drug in plasma samples.


#### 2.4.6. N1-Methylnicotinamide Chloride Reagent (NMNCl)

One mM solution of NMNCl reagent was prepared by dissolving 1.7262 g NMNCl in one liter of distilled water. Aliquot of this solution was diluted with distilled water to obtain5×10-2, 4×10-2, 2×10-2, and 3×10-2 mM solutions.


#### 2.4.7. Sodium Hydroxide Reagent

Sodium hydroxide solutions were prepared in distilled water to have concentration of 0.1 N, 1 N, 2 N, 5 N, and 6 N solutions.


### 2.5. General Fluorometric Procedure

One milliliter of each drug standard solution, assay solution of synthetic mixture, assay solution of pharmaceutical preparation, assay solution of plasma sample, or the assay solution of the volunteer’s plasma was transferred to 10 mL screw capped test tube. Solutions of sodium hydroxide and NMNCl were added. The mixture was cooled in ice for the indicated time then the pH adjusted using formic acid and heated for the indicated time and then was cooled in ice for 5 minutes (optimum NaOH concentration and volume, volume and concentration of added NMNCl, reaction pH values and cooling, and heating times are shown in Table1). The mixture was transferred to 10 mL volumetric flask and the resulting solution was completed using distilled water. The intensity of the resulting fluorescence was measured at the optimal wavelengths indicated in Table 1.Table 1
Optimum conditions for the fluorometric procedure.
DrugpHNaOH conc. (N)NaOH volume (mL)NMNCl conc. (mM)NMNCl volume (mL)Cooling time (min)Heating time (min)λex (nm)λem (nm)14.06.00.73 × 10−20.910328531523.45.01.05 × 10−21.020528431534.11.00.44 × 10−21.015527930643.92.00.62 × 10−20.810428531554.02.00.83 × 10−21.0124280307The fluorometric measurements were performed against reagent blank experiments. Concentrations of the drugs were calculated from the corresponding calibration graphs prepared simultaneously.


## 2.1. Apparatus

Shimadzu RF 5301 PC spectrofluorometer.


## 2.2. Materials

### 2.2.1. Authentic Drugs

Working standards of1,2,3,4, and 5 were supplied by Sigma Pharmaceutical Industries, Egypt, Amyria Pharmaceutical Industries, Alexandria, Egypt, T3A Pharma, Egypt, Medical Union Pharmaceuticals (MUP), Egypt, and Global Napi Pharmaceuticals (GNP), Egypt, respectively.Plasma samples were purchased from the Central Blood Bank of Tanta University Hospital.


### 2.2.2. Other Chemicals

N1-Methylnicotinamide chloride was purchased from Sigma Chemicals Co. Formic acid, sodium hydroxide, methanol, and all other chemicals were of analytical grade. Water used was doubly distilled.


## 2.2.1. Authentic Drugs

Working standards of1,2,3,4, and 5 were supplied by Sigma Pharmaceutical Industries, Egypt, Amyria Pharmaceutical Industries, Alexandria, Egypt, T3A Pharma, Egypt, Medical Union Pharmaceuticals (MUP), Egypt, and Global Napi Pharmaceuticals (GNP), Egypt, respectively.Plasma samples were purchased from the Central Blood Bank of Tanta University Hospital.


## 2.2.2. Other Chemicals

N1-Methylnicotinamide chloride was purchased from Sigma Chemicals Co. Formic acid, sodium hydroxide, methanol, and all other chemicals were of analytical grade. Water used was doubly distilled.


## 2.3. Dosage Forms

### 2.3.1. Sulindac(1)

Rudac tablet (Sigma Pharmaceutical Industries) was labeled to contain 150 and 200 mg, HiDac tablet (Hipharm for Manufactured Pharmaceuticals) labeled to contain 200 mg.


### 2.3.2. Omeprazole(2)

Gastrazole capsules, 20 mg (Amyria Pharmaceutical Industries), Epirazole capsules, 20 mg (Egyptian International Pharmaceutical Industries Co. (EIPICO)), Omepak capsules, 10 and 20 mg (Sedico Pharmaceutical Co.), Pepzole capsules, 40 mg (Alkan Pharma), Gasec capsules, 20 mg (Mepha), Omez capsules, 10 mg (Pharaonia Pharmaceuticals PharoPharma), Gastrocure capsules, 20 mg (October Pharma), Napizole capsules, 20 mg (Global Napi Pharmaceuticals (GNP)), Ulstop capsules, 20 mg (Pharco Pharmaceuticals), Risek capsules, 20 mg (Julphar), Gastroloc capsules, 40 mg (Sigma), Omepral capsules, 20 mg (Memphis Co. for Pharmaceuticals and Chemical Industry), Trio capsules, 20 mg (Alkan Pharma), and Nexium tablets, 20 mg esomeprazole (Astra Pharmaceuticals).


### 2.3.3. Lansoprazole(3)

Lansoprazole capsules, 15 mg (Rexcel), Zollipak capsules, 30 mg (Sedico), Peptazole capsules, 30 mg (Saudi Pharmaceutical Industries and Medical Appliances Corporation (Spimaco), Lanzor capsules, 15 and 30 mg (Aventis Pharma), and Lopral capsules, 30 mg (T3A Pharma).


### 2.3.4. Pantoprazole(4)

Controloc tablets, 40 mg (Byk Gulden Lomberg Chemische Fabrik GmbH), Pantoloc tablets, 20 and 40 mg (Medical Union Pharmaceuticals (MUP)), and Pantazole tablets, 40 mg (Sigma Pharmaceutical Industries).


### 2.3.5. Rabeprazole(5)

Bepra tablets, 20 mg (GNP) and Pariet tablets, 20 mg (Janssen Cilag).All these preparations were purchased from the local market of Egypt except for Nexium that was purchased from Saudi Arabia.


## 2.3.1. Sulindac(1)

Rudac tablet (Sigma Pharmaceutical Industries) was labeled to contain 150 and 200 mg, HiDac tablet (Hipharm for Manufactured Pharmaceuticals) labeled to contain 200 mg.


## 2.3.2. Omeprazole(2)

Gastrazole capsules, 20 mg (Amyria Pharmaceutical Industries), Epirazole capsules, 20 mg (Egyptian International Pharmaceutical Industries Co. (EIPICO)), Omepak capsules, 10 and 20 mg (Sedico Pharmaceutical Co.), Pepzole capsules, 40 mg (Alkan Pharma), Gasec capsules, 20 mg (Mepha), Omez capsules, 10 mg (Pharaonia Pharmaceuticals PharoPharma), Gastrocure capsules, 20 mg (October Pharma), Napizole capsules, 20 mg (Global Napi Pharmaceuticals (GNP)), Ulstop capsules, 20 mg (Pharco Pharmaceuticals), Risek capsules, 20 mg (Julphar), Gastroloc capsules, 40 mg (Sigma), Omepral capsules, 20 mg (Memphis Co. for Pharmaceuticals and Chemical Industry), Trio capsules, 20 mg (Alkan Pharma), and Nexium tablets, 20 mg esomeprazole (Astra Pharmaceuticals).


## 2.3.3. Lansoprazole(3)

Lansoprazole capsules, 15 mg (Rexcel), Zollipak capsules, 30 mg (Sedico), Peptazole capsules, 30 mg (Saudi Pharmaceutical Industries and Medical Appliances Corporation (Spimaco), Lanzor capsules, 15 and 30 mg (Aventis Pharma), and Lopral capsules, 30 mg (T3A Pharma).


## 2.3.4. Pantoprazole(4)

Controloc tablets, 40 mg (Byk Gulden Lomberg Chemische Fabrik GmbH), Pantoloc tablets, 20 and 40 mg (Medical Union Pharmaceuticals (MUP)), and Pantazole tablets, 40 mg (Sigma Pharmaceutical Industries).


## 2.3.5. Rabeprazole(5)

Bepra tablets, 20 mg (GNP) and Pariet tablets, 20 mg (Janssen Cilag).All these preparations were purchased from the local market of Egypt except for Nexium that was purchased from Saudi Arabia.


## 2.4. Reagents and Standard Solutions

### 2.4.1. Stock Standard Solutions of Drugs

Stock standard solutions were prepared in 0.1 N sodium hydroxide for1, 2, and 4; 50% aqueous methanol for 3 and methanol for 5 to contain 100 mg/mL for 1, 150 μg/mL for 3, 4, and 5, and 250 μg/mL for 5.


### 2.4.2. Serial Standard Solutions of Drugs

Aliquots of the stock solutions were diluted quantitatively with the same solvent to obtain concentration ranging between 10–500μg/mL for 1, 0.1–15 μg/mL for 2, 3, and 4, and 0.1–25 μg/mL for 5.


### 2.4.3. Assay Solutions of Drugs in Synthetic Mixtures

Several synthetic mixtures were prepared for1, 4, and 5 to contain different proportions of the possible interfering substances that may be present with the drug in its dosage form. Three synthetic mixtures containing 1 were prepared. The first mixture contained 200 mg 1, 230 mg cellulose, 10 mg magnesium stearate, and 60 mg starch. The second mixture contained 200 mg 1, 100 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 130 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing4along with various additives and inactive ingredients were prepared. The first mixture contained 20 mg 4, 280 mg avicel, 60 mg maize starch, 10 mg magnesium stearate, 10 mg magnesium carbonate, 1 mg quinolone, 1 mg acidisol, 60 mg talc, 1 mg colloidal silicon, 6 mg polyethylene glycol 6000, 50 mg Eudragit L100-55, and 1 mg titanium dioxide. The second mixture contained 20 mg 4, 280 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 340 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing5 were prepared. The first mixture contained 20 mg 5, 300 mg croscarmellose sodium, 72 mg talc, 1 mg sodium hydroxide, 1 mg titanium dioxide, and 8 mg magnesium stearate. The second mixture contained 20 mg 5, 180 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 240 mg avicel instead of lactose and gelatin.Each synthetic mixture of1 and 4 was extracted with 100 mL 0.1 N sodium hydroxide. The synthetic mixture of 5 was extracted with 100 mL methanol. The extracts were filtered, and the first 10 mL of the filtrate were rejected. Aliquots of the filtrate were diluted with the same solvent to obtain serial solutions in the concentration ranges 10–500 μg/mL, 0.1–15 μg/mL, and 0.1–25 μg/mL for 1, 4, and 5, respectively.


### 2.4.4. Assay Solutions of Drugs in Their Pharmaceutical Preparations

A quantity of the mixed contents of 20 capsules or tablets equivalent to one capsule or tablet of1–5 were finely powdered and transferred with the aid of several portions of 0.1 N sodium hydroxide solution (for 1, 2, and 4), 50% aqueous methanol (for 3), or methanol (for 5) to a 100 mL volumetric flask, and the volume was completed with the same solvent. The resulting solution was filtered and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain 200 μg/mL, 6 μg/mL, 3 μg/mL, 4 μg/mL, and 6 μg/mL solutions of 1–5, respectively.


### 2.4.5. Assay Solutions of Drugs in Spiked Human Plasma Samples

Serial Standard Solutions of the Drugs
Serial standard solutions for1,2,3, and 4 were prepared in 0.1 N sodium hydroxide solution in concentrations ranging between 1–50 mg/mL for 1and 50–1500 μg/mL for 2,3, and 4. Serial standard solutions of 5 were prepared in methanol in concentrations ranges of 1–2500 μg/mL.Preparation of Spiked Human Plasma Samples
Two hundredμL of each 1,2,3, and 4 serial standard solution were diluted with 1800 μL human plasma and vortex mixed to obtain concentrations ranging between 0.1–5 mg/mL for 1 and 5–150 μg/mL for 2,3, and 4. For 5 200 μL of each serial standard solution were evaporated, the residue was dissolved in 1800 μL human plasma and vortex mixed, and 200 μL distilled water were added and vortex mixed to obtain 1–250 μg/mL.Preparation of Assay Solutions of Drugs in Plasma Samples
Two hundredμL of each spiked human plasma samples were mixed with 1800 μL methanol and centrifuged for 15 minutes to separate the precipitated protein. The clear supernatant was filtered through Millipore filter (0.45 μm) to obtain solutions in concentration ranging between 10–500 μg/mL for 1, 0.5–15 μg/mL for 2, 3, and 4, and 0.1–25 μg/mL for 5.Determination of1–5 in Volunteer’s Blood or Hospitalized Patient’s Blood
A blood sample was withdrawn in a test tube to which heparin was previously added and dried. The sample was centrifuged to separate plasma and then treated as previously mentioned under preparation of assay solutions of each drug in plasma samples.


### 2.4.6. N1-Methylnicotinamide Chloride Reagent (NMNCl)

One mM solution of NMNCl reagent was prepared by dissolving 1.7262 g NMNCl in one liter of distilled water. Aliquot of this solution was diluted with distilled water to obtain5×10-2, 4×10-2, 2×10-2, and 3×10-2 mM solutions.


### 2.4.7. Sodium Hydroxide Reagent

Sodium hydroxide solutions were prepared in distilled water to have concentration of 0.1 N, 1 N, 2 N, 5 N, and 6 N solutions.


## 2.4.1. Stock Standard Solutions of Drugs

Stock standard solutions were prepared in 0.1 N sodium hydroxide for1, 2, and 4; 50% aqueous methanol for 3 and methanol for 5 to contain 100 mg/mL for 1, 150 μg/mL for 3, 4, and 5, and 250 μg/mL for 5.


## 2.4.2. Serial Standard Solutions of Drugs

Aliquots of the stock solutions were diluted quantitatively with the same solvent to obtain concentration ranging between 10–500μg/mL for 1, 0.1–15 μg/mL for 2, 3, and 4, and 0.1–25 μg/mL for 5.


## 2.4.3. Assay Solutions of Drugs in Synthetic Mixtures

Several synthetic mixtures were prepared for1, 4, and 5 to contain different proportions of the possible interfering substances that may be present with the drug in its dosage form. Three synthetic mixtures containing 1 were prepared. The first mixture contained 200 mg 1, 230 mg cellulose, 10 mg magnesium stearate, and 60 mg starch. The second mixture contained 200 mg 1, 100 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 130 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing4along with various additives and inactive ingredients were prepared. The first mixture contained 20 mg 4, 280 mg avicel, 60 mg maize starch, 10 mg magnesium stearate, 10 mg magnesium carbonate, 1 mg quinolone, 1 mg acidisol, 60 mg talc, 1 mg colloidal silicon, 6 mg polyethylene glycol 6000, 50 mg Eudragit L100-55, and 1 mg titanium dioxide. The second mixture contained 20 mg 4, 280 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 340 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing5 were prepared. The first mixture contained 20 mg 5, 300 mg croscarmellose sodium, 72 mg talc, 1 mg sodium hydroxide, 1 mg titanium dioxide, and 8 mg magnesium stearate. The second mixture contained 20 mg 5, 180 mg lactose, 60 mg starch, 60 mg gelatin, 8 mg magnesium stearate, and 72 mg talc. The third mixture contained 240 mg avicel instead of lactose and gelatin.Each synthetic mixture of1 and 4 was extracted with 100 mL 0.1 N sodium hydroxide. The synthetic mixture of 5 was extracted with 100 mL methanol. The extracts were filtered, and the first 10 mL of the filtrate were rejected. Aliquots of the filtrate were diluted with the same solvent to obtain serial solutions in the concentration ranges 10–500 μg/mL, 0.1–15 μg/mL, and 0.1–25 μg/mL for 1, 4, and 5, respectively.


## 2.4.4. Assay Solutions of Drugs in Their Pharmaceutical Preparations

A quantity of the mixed contents of 20 capsules or tablets equivalent to one capsule or tablet of1–5 were finely powdered and transferred with the aid of several portions of 0.1 N sodium hydroxide solution (for 1, 2, and 4), 50% aqueous methanol (for 3), or methanol (for 5) to a 100 mL volumetric flask, and the volume was completed with the same solvent. The resulting solution was filtered and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain 200 μg/mL, 6 μg/mL, 3 μg/mL, 4 μg/mL, and 6 μg/mL solutions of 1–5, respectively.


## 2.4.5. Assay Solutions of Drugs in Spiked Human Plasma Samples

Serial Standard Solutions of the Drugs
Serial standard solutions for1,2,3, and 4 were prepared in 0.1 N sodium hydroxide solution in concentrations ranging between 1–50 mg/mL for 1and 50–1500 μg/mL for 2,3, and 4. Serial standard solutions of 5 were prepared in methanol in concentrations ranges of 1–2500 μg/mL.Preparation of Spiked Human Plasma Samples
Two hundredμL of each 1,2,3, and 4 serial standard solution were diluted with 1800 μL human plasma and vortex mixed to obtain concentrations ranging between 0.1–5 mg/mL for 1 and 5–150 μg/mL for 2,3, and 4. For 5 200 μL of each serial standard solution were evaporated, the residue was dissolved in 1800 μL human plasma and vortex mixed, and 200 μL distilled water were added and vortex mixed to obtain 1–250 μg/mL.Preparation of Assay Solutions of Drugs in Plasma Samples
Two hundredμL of each spiked human plasma samples were mixed with 1800 μL methanol and centrifuged for 15 minutes to separate the precipitated protein. The clear supernatant was filtered through Millipore filter (0.45 μm) to obtain solutions in concentration ranging between 10–500 μg/mL for 1, 0.5–15 μg/mL for 2, 3, and 4, and 0.1–25 μg/mL for 5.Determination of1–5 in Volunteer’s Blood or Hospitalized Patient’s Blood
A blood sample was withdrawn in a test tube to which heparin was previously added and dried. The sample was centrifuged to separate plasma and then treated as previously mentioned under preparation of assay solutions of each drug in plasma samples.


## 2.4.6. N1-Methylnicotinamide Chloride Reagent (NMNCl)

One mM solution of NMNCl reagent was prepared by dissolving 1.7262 g NMNCl in one liter of distilled water. Aliquot of this solution was diluted with distilled water to obtain5×10-2, 4×10-2, 2×10-2, and 3×10-2 mM solutions.


## 2.4.7. Sodium Hydroxide Reagent

Sodium hydroxide solutions were prepared in distilled water to have concentration of 0.1 N, 1 N, 2 N, 5 N, and 6 N solutions.


## 2.5. General Fluorometric Procedure

One milliliter of each drug standard solution, assay solution of synthetic mixture, assay solution of pharmaceutical preparation, assay solution of plasma sample, or the assay solution of the volunteer’s plasma was transferred to 10 mL screw capped test tube. Solutions of sodium hydroxide and NMNCl were added. The mixture was cooled in ice for the indicated time then the pH adjusted using formic acid and heated for the indicated time and then was cooled in ice for 5 minutes (optimum NaOH concentration and volume, volume and concentration of added NMNCl, reaction pH values and cooling, and heating times are shown in Table1). The mixture was transferred to 10 mL volumetric flask and the resulting solution was completed using distilled water. The intensity of the resulting fluorescence was measured at the optimal wavelengths indicated in Table 1.Table 1
Optimum conditions for the fluorometric procedure.
DrugpHNaOH conc. (N)NaOH volume (mL)NMNCl conc. (mM)NMNCl volume (mL)Cooling time (min)Heating time (min)λex (nm)λem (nm)14.06.00.73 × 10−20.910328531523.45.01.05 × 10−21.020528431534.11.00.44 × 10−21.015527930643.92.00.62 × 10−20.810428531554.02.00.83 × 10−21.0124280307The fluorometric measurements were performed against reagent blank experiments. Concentrations of the drugs were calculated from the corresponding calibration graphs prepared simultaneously.


## 3. Results and Discussion

The reaction of NMNCl, with drugs containing active methylene sulfoxide groups, was not previously studied and this is the first application of this reaction to compounds containing this functional group. When1–5 (for chemical structures, cf. Figure 1 and for plausible chemical pathway, cf. [4]) were allowed to react with NMNCl under the optimal conditions specified for each, strong fluorescent products were produced except for 1 that was found to have a quenching effect on the fluorescence of the reagent blank used at excitation and emission wavelengths of 285 nm and 315 nm, respectively; compare Figure 2. The optimal wavelengths of excitation and emission of the reaction product were determined using synchronous wavelength search compare Table 1.Figure 1
Chemical structures of1–5.Figure 2
Excitation and emission spectra of the reaction product of1with NMNCl.Different variables affecting the reaction between the estimated drug and NMNCl, including sodium hydroxide concentration and volume, added NMNCl concentration and volume, cooling time, heating time, and pH variations, were studied to optimize the reaction conditions to give maximum fluorescence intensity; compare Figures3, 4, 5, 6, 7, 8, and 9.Effect of NaOH concentration (N) on fluorescence intensity of the reaction products of1–5 with NMNCl. The variation of NaOH concentration is made at constant volume.
(a)(b)Effect of NaOH volume (mL) on fluorescence intensity of reaction product of1–5 with NMNCl. The variation of NaOH volume is made at constant concentration.
(a)(b)Effect of NMNCl concentration (mM) on fluorescence intensity of reaction product of1–5 with NMNCl. The variation of NMNCl concentration is made at constant volume.
(a)(b)Effect of NMNCl volume (mL) on fluorescence intensity of reaction product of1–5 with NMNCl. The variation of NMNCl volume is made at constant concentration.
(a)(b)Effect of cooling time on fluorescence intensity of reaction product of1–5 with NMNCl.
(a)(b)Effect of heating time on fluorescence intensity of reaction product of1–5 with NMNCl.
(a)(b)Effect of pH on fluorescence intensity of each reaction product of1–5 with NMNCl.
(a)(b)After optimization of the different reaction parameters, linear relationships between fluorescence intensity and each drug concentration were obtained over the following concentration ranging between 50–1200 ng/mL, 100–1500 ng/mL, 100–1500 ng/mL, and 20–2200 ng/mL in the standard solutions of2, 3, 4, and 5, respectively, and 75–1200 ng/mL, 100–1500 ng/mL, 100–1400 ng/mL and, 50–2100 ng/mL in plasma samples for 2,3,4, and5, respectively. The linearity range was 1–50 μg/mL in both standard solutions and spiked human plasma samples for the observed fluorescence quenching produced by 1.These results reveal a good and dynamic linearity ranges of the proposed method with different drugs. The good linearity of these relations was indicated by the corresponding regression equations shown in Tables2 and 3, for standard solutions and spiked human plasma samples, respectively.Table 2
Regression analysis parameters for the determination of1–5 in standard solutions using the proposed method.
DrugLinearity rangeSlope*Intercept*R2MeanSEMeanSE11–50μg/mL4.0780.0242.750.390.998250–1200 ng/mL0.66940.000515.2261.3760.99973100–1500 ng/mL0.51040.0199.8263.3760.9983410–1500 ng/mL0.62150.05573.2740.2980.999520–2200 ng/mL0.4360.0213.311.550.9998Average of triplicate analyses, 15 calibration data points.* Three calibrations curves were performed for each drug.Table 3
Regression analysis parameters for the determination of1–5 in spiked human plasma samples using the proposed method.
DrugLinearity rangeSlope*Intercept*R2MeanSEMeanSE11–50μg/mL4.07710.02342.6740.780.994275–1200 ng/mL0.66730.00616.39352.1680.99493100–1400 ng/mL0.50840.0035100.7310.0190.9971410–1500 ng/mL0.62830.1267.4190.390.9989550–2100 ng/mL0.4660.0110.31.250.999Average of triplicate analyses, 15 calibration data points.*Three calibrations curves were performed for each drug.


### 3.1. Detection Limit (DL)

Detection limits were practically determined according to the ICH topic Q2B (R1) [31] and found to be 0.5 μg/mL, 10 ng/mL, 20 ng/mL, 20 ng/mL, and 1 ng/mL in standard solutions for 1,2,3,4, and 5, respectively, and 0.5 μg/mL, 25 ng/mL, 30 ng/mL, 50 ng/mL, and 10 ng/mL in plasma samples for 1,2,3,4, and 5, respectively.


### 3.2. Quantitation Limit (QL)

Quantitation limits were practically determined according to the ICH topic Q2B (R1) [31] and found to be 1 μg/mL, 50 ng/mL, 100 ng/mL, 100 ng/mL, and 20 ng/mL in standard solutions for 1,2,3,4, and 5, respectively, and 1 μg/mL, 75 ng/mL, 100 ng/mL, 100 ng/mL and 50 ng/mL in plasma samples for 1,2,3,4 and 5, respectively. These results show the high sensitivity of the proposed method.


### 3.3. Accuracy

The accuracy of the proposed method was studied according to the ICH topic Q2B (R1) [31], by preparing standard solutions and spiked human plasma samples containing various concentrations, lying within the linearity range of each drug, and analyzing them using the proposed method. The results, expressed as % recovery ± S.D., are shown in Tables 4 and 5 for standard solutions and spiked human plasma samples, respectively.Table 4
Recovery data of1–5 when assayed in standard solutions using the proposed method.
DrugClaimed drug concentrationRecovered concentration*% recoveryMean % recovery ± S.D.CV1μg/mL13 12 15 20 30 40 500.993.01 12.02 14.99 20.1 30 39.99 49.8999%100.3% 100.16% 99.9% 100.5% 100% 99.9% 99.8%99.4±0.440.45%2ng/mL5075 200 500 1000 120050.174.4 203 500 1004 1199100.2%99.2% 101.5% 100% 100.4% 99.9%100.2±0.750.755%3ng/mL100300 1200 150098301 1195 150998%100.3% 99.6% 100.6%99.625±1.161.166%4ng/mL100300 400 600 800 1000 1200 150099.5301 403 597 799 1002 1198 149399.5%100.3% 100.75% 99.5% 99.87% 100.2% 99.83% 99.53%99.94±0.460.455%5ng/mL20200 600 800 1000 1500 220020.2202 606 795 990 1510 2185101%101% 101% 99.4% 99% 100.6% 99.3%100.5±0.920.92%*Average of triplicate analyses for each concentration.Table 5
Recovery data of1–5 when assayed in spiked human plasma samples using the proposed method.
DrugClaimed drug concentrationRecovered concentration*% recoveryMean % recovery ± S.D.CV1μg/mL13 12 15 20 30 40 501.012.9 12.02 15.1 19.89 30.15 40.09 49.9101%97.5% 100.15% 100.6% 99.45% 100.5% 100.3% 99.8%99.98±1.041.05%2ng/mL75200 500 1000 120073.5210 500 9700 122498%105% 100% 97% 102%104±3.213.20%3ng/mL100300 1200 1500103295 1204 1498103%98.3% 100.3% 99.8%100.26±1.711.72%4ng/mL100300 400 600 800 1000 1200 1400101302 399 603 795 995 1204 1392101%100.67% 99.75% 100.5% 99.375% 99.5% 100.33% 99.4%100.11±0.610.62%5ng/mL50200 600 800 1000 1500 210051196 602 810 1012 1485 2125102%98% 100.3% 101.25% 101.2% 99% 101.2%100.42±431.43%*Average of triplicate analyses for each concentration.


### 3.4. Precision

The precision of the method was judged by performing intraday and interday triplicate analyses of different concentrations covering the linearity range of each drug in both standard solutions and spiked human plasma samples. The results are reported as S.D. and C.V. in Tables6 and 7 for standard solutions and spiked human plasma samples, respectively.Table 6
Intraday and interday precision data of 1–5 in standard solutions using the proposed method.
DrugClaimed conc.IntradayInterdayFound conc.*S.D.C.V.Found conc.*S.D.C.V.1μg/mL10.9950.0350.354%0.990.0070.71%33.010.0070.235%2.980.0140.473%1211.990.0070.058%12.020.0140.118%1515.010.0070.047%15.0250.0180.118%2019.980.0140.071%20.010.0070.035%3030.0150.0110.035%29.990.0070.024%4039.950.0350.088%39.980.0140.036%5049.7150.2020.404%50.010.0070.014%2ng/mL5050.10.070.14%49.80. 140.28%3203221.40.44%3200.00.0%600600.10.070.012%6020.140.24%115011510.710.615%11453.5350.308%120011981.410.1179%12032.1210.176%3ng/mL1001010.710.703%990.711.4%3002990.710.24%2981.410.47%120012021.1410.118%12021.1410.118%130012981.410.11%13032.1210.163%150014990.710.47%15042.830.188%4ng/mL1001010.7070.704%990.7070.71%3003021.4140.469%3032.120.703%4003990.7070.177%4021.4140.353%6005981.4140.236%5972.120.354%8007981.4140.176%7990.710.088%100010042.8280.282%10053.540.353%120011953.530.295%12010.710.059%150014972.120.142%15042.830.188%5ng/mL2020.20.140.7%19.70.211.07%2002010.710.35%2021.410.71%6006053.50.6%5962.830.47%8007916.360.8%8064.30.53%10009907.10.71%10128.50.84%150014907.10.47151812.80.84%2000198510.60.53%20096.40.32%2200222014.140.64%21907.10.15%*Average of triplicate analyses for each concentration.Table 7
Intraday and interday precision data of1–5 in plasma samples using the proposed method.
DrugClaimed conc.IntradayInterdayFound conc.*S.D.C.V.Found conc.*S.D.C.V.1μg/mL10.960.282.88%0.960.0282.88%21.980.0140.71%2.050.0351.76%1010.10.0710.704%9.950.0350.35%1313.150.1060.811%12.80.1411.09%1515.20.1410.936%14.80.1410.949%2019.90.0710.354%20.20.1410.704%3029.80.1410.473%30.20.1410.47%4040.20.1410.353%39.750.1770.44%5050.10.0710.141%49.70.2120.43%2ng/mL12001219141.11%12000.000.0%100098890.86%10201.41.4%5004971.43%50111.2%20019073.6%20642.1%757410.95%72.522.4%3ng/mL1001021.4141.4%1010.710.704%3003053.5361.169%2981.1410.47%120011953.5360.245%12064.250.353%130013010.710.0544%13042.830.217%150015021.4140.094%15010.710.047%4ng/mL100100.50.3540.353%1010.7070.704%3003021.14140.469%2990.7070.236%4003981.14140.354%4032.120.528%6005972.120.354%6010.7070.118%8008042.8280.353%7944.230.532%100010053.5360.353%9953.5360.354%120011944.240.354%12053.5360.294%130013042.8282.17%13042.8282.17%140014085.650.402%13962.830.202%5ng/mL50490.711.43%50.20.140.28%2001990.710.35%2032.121.1%6006085.660.93%5953.530.6%8008107.10.9%7907.10.88%1000101510.61.1%9907.10.71%1500152014.10.94%151510.60.71%1800178510.60.6%178113.40.75%2100211510.60.5%20953.50.16%*Average of triplicate analyses for each concentration.


### 3.5. Specificity

To study the method specificity, three synthetic mixtures of1,4, and 5 were prepared to contain the possible interfering substances used during pharmaceutical formulations. These mixtures were analyzed using the proposed method and the results, expressed as % recovery ± S.D. No synthetic mixtures were prepared for 2 or 3 because these drugs are supplied as capsules containing the active ingredient without additives. The prepared mixtures were determined by the proposed method and the results, expressed as % recovery ± S.D., were found to be 99.58%±3.47, 99.3%±0.9, and 100.5%±0.67, for 1,4, and 5, respectively.


### 3.6. Assay of Pharmaceutical Preparations

All the pharmaceutical preparations available in the local market for each drug were analyzed using the proposed method. The results, expressed as % recovery ± S.D., are illustrated in Table8.Table 8
Results of the proposed method recovery experiments of1–5 of different pharmaceutical preparations.
DrugPharmaceutical preparations% recovery ± S.D.*1Rudac 150 mg tablet102±0.56%Rudac 200 mg tablet101±2.1%HiDac 200 mg tablet98.5±1.55%2Epirazole 20 mg capsule100.1±1.37%Gastrozole 20 mg capsule100±0.95%Gastrocure 20 mg capsule99±1.22%Napizole 20 mg capsule98.45±0.88%Omepak 20 mg capsule99.98±1.56%Omepak 10 mg capsule100±1.11%Ulstop 20 mg capsule97.9±0.99%Gasec 20 mg capsule100.2±1.52%Risek 20 mg capsule98±1.63%Pepzole 40 mg capsule97.88±0.54%Gastroloc 40 mg capsule97.2±0.55%Omepral 20 mg capsule99±1.42%Omez 10 mg capsule99.9±0.78%Trio 20 mg capsule98.1±1.74%Nexium 20 mg (esomeprazole magnesium tablet)**96.12±0.56%3Lansoprazole 30 mg cap100.6±2.31%Zollipak 30 mg cap101±1.51%Peptazole 30 mg cap99±2.1%Lanzor 15 mg cap100.1±1.2%Lanzor 30 mg cap99.8±0.95%Lopral30 mg cap99±0.89%4Controloc 20 mg tablet98±1.98%Pantoloc 20 mg tablet97±0.51%Pantoloc 40 mg tablet101±1.12%Pantazole 40 mg tablet101.5±0.52%5Bepra 20 mg tablet102±1.35%Pariet 20 mg tablet100±2.1%*Average of triplicate analyses.** The (S)-enantiomer of omeprazole; the pharmacologically active form.


### 3.7. Determination of1–5 in Hospitalized Patient’s or Volunteer’s Blood

The success in the application of the highly sensitive proposed procedure for the determination of1–5 in spiked human plasma samples with good accuracy and precision encouraged the investigators to study its application for monitoring the drug level in the blood of a volunteer or a hospitalized patient under 1–5 therapy.The level of2 was monitored in the blood of a hospitalized patient receiving it with other medications as paracetamol, bromohexine HCl, acephylline piperazine, hyosine N-butylbromide, and captopril. The concentration of 2 in the patient’s blood was found to be 0.31 μg/mL.The level of each of1,3,4, and 5 was monitored in the blood of volunteers. The concentrations of 1,3,4, and 5 in volunteers’ blood were found to be 5.2 μg/mL, 1.1 μg/mL, 3.6 μg/mL, and 0.3 μg/mL, respectively.


## 3.1. Detection Limit (DL)

Detection limits were practically determined according to the ICH topic Q2B (R1) [31] and found to be 0.5 μg/mL, 10 ng/mL, 20 ng/mL, 20 ng/mL, and 1 ng/mL in standard solutions for 1,2,3,4, and 5, respectively, and 0.5 μg/mL, 25 ng/mL, 30 ng/mL, 50 ng/mL, and 10 ng/mL in plasma samples for 1,2,3,4, and 5, respectively.


## 3.2. Quantitation Limit (QL)

Quantitation limits were practically determined according to the ICH topic Q2B (R1) [31] and found to be 1 μg/mL, 50 ng/mL, 100 ng/mL, 100 ng/mL, and 20 ng/mL in standard solutions for 1,2,3,4, and 5, respectively, and 1 μg/mL, 75 ng/mL, 100 ng/mL, 100 ng/mL and 50 ng/mL in plasma samples for 1,2,3,4 and 5, respectively. These results show the high sensitivity of the proposed method.


## 3.3. Accuracy

The accuracy of the proposed method was studied according to the ICH topic Q2B (R1) [31], by preparing standard solutions and spiked human plasma samples containing various concentrations, lying within the linearity range of each drug, and analyzing them using the proposed method. The results, expressed as % recovery ± S.D., are shown in Tables 4 and 5 for standard solutions and spiked human plasma samples, respectively.Table 4
Recovery data of1–5 when assayed in standard solutions using the proposed method.
DrugClaimed drug concentrationRecovered concentration*% recoveryMean % recovery ± S.D.CV1μg/mL13 12 15 20 30 40 500.993.01 12.02 14.99 20.1 30 39.99 49.8999%100.3% 100.16% 99.9% 100.5% 100% 99.9% 99.8%99.4±0.440.45%2ng/mL5075 200 500 1000 120050.174.4 203 500 1004 1199100.2%99.2% 101.5% 100% 100.4% 99.9%100.2±0.750.755%3ng/mL100300 1200 150098301 1195 150998%100.3% 99.6% 100.6%99.625±1.161.166%4ng/mL100300 400 600 800 1000 1200 150099.5301 403 597 799 1002 1198 149399.5%100.3% 100.75% 99.5% 99.87% 100.2% 99.83% 99.53%99.94±0.460.455%5ng/mL20200 600 800 1000 1500 220020.2202 606 795 990 1510 2185101%101% 101% 99.4% 99% 100.6% 99.3%100.5±0.920.92%*Average of triplicate analyses for each concentration.Table 5
Recovery data of1–5 when assayed in spiked human plasma samples using the proposed method.
DrugClaimed drug concentrationRecovered concentration*% recoveryMean % recovery ± S.D.CV1μg/mL13 12 15 20 30 40 501.012.9 12.02 15.1 19.89 30.15 40.09 49.9101%97.5% 100.15% 100.6% 99.45% 100.5% 100.3% 99.8%99.98±1.041.05%2ng/mL75200 500 1000 120073.5210 500 9700 122498%105% 100% 97% 102%104±3.213.20%3ng/mL100300 1200 1500103295 1204 1498103%98.3% 100.3% 99.8%100.26±1.711.72%4ng/mL100300 400 600 800 1000 1200 1400101302 399 603 795 995 1204 1392101%100.67% 99.75% 100.5% 99.375% 99.5% 100.33% 99.4%100.11±0.610.62%5ng/mL50200 600 800 1000 1500 210051196 602 810 1012 1485 2125102%98% 100.3% 101.25% 101.2% 99% 101.2%100.42±431.43%*Average of triplicate analyses for each concentration.


## 3.4. Precision

The precision of the method was judged by performing intraday and interday triplicate analyses of different concentrations covering the linearity range of each drug in both standard solutions and spiked human plasma samples. The results are reported as S.D. and C.V. in Tables6 and 7 for standard solutions and spiked human plasma samples, respectively.Table 6
Intraday and interday precision data of 1–5 in standard solutions using the proposed method.
DrugClaimed conc.IntradayInterdayFound conc.*S.D.C.V.Found conc.*S.D.C.V.1μg/mL10.9950.0350.354%0.990.0070.71%33.010.0070.235%2.980.0140.473%1211.990.0070.058%12.020.0140.118%1515.010.0070.047%15.0250.0180.118%2019.980.0140.071%20.010.0070.035%3030.0150.0110.035%29.990.0070.024%4039.950.0350.088%39.980.0140.036%5049.7150.2020.404%50.010.0070.014%2ng/mL5050.10.070.14%49.80. 140.28%3203221.40.44%3200.00.0%600600.10.070.012%6020.140.24%115011510.710.615%11453.5350.308%120011981.410.1179%12032.1210.176%3ng/mL1001010.710.703%990.711.4%3002990.710.24%2981.410.47%120012021.1410.118%12021.1410.118%130012981.410.11%13032.1210.163%150014990.710.47%15042.830.188%4ng/mL1001010.7070.704%990.7070.71%3003021.4140.469%3032.120.703%4003990.7070.177%4021.4140.353%6005981.4140.236%5972.120.354%8007981.4140.176%7990.710.088%100010042.8280.282%10053.540.353%120011953.530.295%12010.710.059%150014972.120.142%15042.830.188%5ng/mL2020.20.140.7%19.70.211.07%2002010.710.35%2021.410.71%6006053.50.6%5962.830.47%8007916.360.8%8064.30.53%10009907.10.71%10128.50.84%150014907.10.47151812.80.84%2000198510.60.53%20096.40.32%2200222014.140.64%21907.10.15%*Average of triplicate analyses for each concentration.Table 7
Intraday and interday precision data of1–5 in plasma samples using the proposed method.
DrugClaimed conc.IntradayInterdayFound conc.*S.D.C.V.Found conc.*S.D.C.V.1μg/mL10.960.282.88%0.960.0282.88%21.980.0140.71%2.050.0351.76%1010.10.0710.704%9.950.0350.35%1313.150.1060.811%12.80.1411.09%1515.20.1410.936%14.80.1410.949%2019.90.0710.354%20.20.1410.704%3029.80.1410.473%30.20.1410.47%4040.20.1410.353%39.750.1770.44%5050.10.0710.141%49.70.2120.43%2ng/mL12001219141.11%12000.000.0%100098890.86%10201.41.4%5004971.43%50111.2%20019073.6%20642.1%757410.95%72.522.4%3ng/mL1001021.4141.4%1010.710.704%3003053.5361.169%2981.1410.47%120011953.5360.245%12064.250.353%130013010.710.0544%13042.830.217%150015021.4140.094%15010.710.047%4ng/mL100100.50.3540.353%1010.7070.704%3003021.14140.469%2990.7070.236%4003981.14140.354%4032.120.528%6005972.120.354%6010.7070.118%8008042.8280.353%7944.230.532%100010053.5360.353%9953.5360.354%120011944.240.354%12053.5360.294%130013042.8282.17%13042.8282.17%140014085.650.402%13962.830.202%5ng/mL50490.711.43%50.20.140.28%2001990.710.35%2032.121.1%6006085.660.93%5953.530.6%8008107.10.9%7907.10.88%1000101510.61.1%9907.10.71%1500152014.10.94%151510.60.71%1800178510.60.6%178113.40.75%2100211510.60.5%20953.50.16%*Average of triplicate analyses for each concentration.


## 3.5. Specificity

To study the method specificity, three synthetic mixtures of1,4, and 5 were prepared to contain the possible interfering substances used during pharmaceutical formulations. These mixtures were analyzed using the proposed method and the results, expressed as % recovery ± S.D. No synthetic mixtures were prepared for 2 or 3 because these drugs are supplied as capsules containing the active ingredient without additives. The prepared mixtures were determined by the proposed method and the results, expressed as % recovery ± S.D., were found to be 99.58%±3.47, 99.3%±0.9, and 100.5%±0.67, for 1,4, and 5, respectively.


## 3.6. Assay of Pharmaceutical Preparations

All the pharmaceutical preparations available in the local market for each drug were analyzed using the proposed method. The results, expressed as % recovery ± S.D., are illustrated in Table8.Table 8
Results of the proposed method recovery experiments of1–5 of different pharmaceutical preparations.
DrugPharmaceutical preparations% recovery ± S.D.*1Rudac 150 mg tablet102±0.56%Rudac 200 mg tablet101±2.1%HiDac 200 mg tablet98.5±1.55%2Epirazole 20 mg capsule100.1±1.37%Gastrozole 20 mg capsule100±0.95%Gastrocure 20 mg capsule99±1.22%Napizole 20 mg capsule98.45±0.88%Omepak 20 mg capsule99.98±1.56%Omepak 10 mg capsule100±1.11%Ulstop 20 mg capsule97.9±0.99%Gasec 20 mg capsule100.2±1.52%Risek 20 mg capsule98±1.63%Pepzole 40 mg capsule97.88±0.54%Gastroloc 40 mg capsule97.2±0.55%Omepral 20 mg capsule99±1.42%Omez 10 mg capsule99.9±0.78%Trio 20 mg capsule98.1±1.74%Nexium 20 mg (esomeprazole magnesium tablet)**96.12±0.56%3Lansoprazole 30 mg cap100.6±2.31%Zollipak 30 mg cap101±1.51%Peptazole 30 mg cap99±2.1%Lanzor 15 mg cap100.1±1.2%Lanzor 30 mg cap99.8±0.95%Lopral30 mg cap99±0.89%4Controloc 20 mg tablet98±1.98%Pantoloc 20 mg tablet97±0.51%Pantoloc 40 mg tablet101±1.12%Pantazole 40 mg tablet101.5±0.52%5Bepra 20 mg tablet102±1.35%Pariet 20 mg tablet100±2.1%*Average of triplicate analyses.** The (S)-enantiomer of omeprazole; the pharmacologically active form.


## 3.7. Determination of1–5 in Hospitalized Patient’s or Volunteer’s Blood

The success in the application of the highly sensitive proposed procedure for the determination of1–5 in spiked human plasma samples with good accuracy and precision encouraged the investigators to study its application for monitoring the drug level in the blood of a volunteer or a hospitalized patient under 1–5 therapy.The level of2 was monitored in the blood of a hospitalized patient receiving it with other medications as paracetamol, bromohexine HCl, acephylline piperazine, hyosine N-butylbromide, and captopril. The concentration of 2 in the patient’s blood was found to be 0.31 μg/mL.The level of each of1,3,4, and 5 was monitored in the blood of volunteers. The concentrations of 1,3,4, and 5 in volunteers’ blood were found to be 5.2 μg/mL, 1.1 μg/mL, 3.6 μg/mL, and 0.3 μg/mL, respectively.


## 4. Conclusion

The proposed method makes use of the high sensitivity and specificity of the fluorometric analysis to reach low limits of detection and quantitation for all the studied drugs in standard solutions, synthetic mixtures, pharmaceutical preparations, spiked human plasma samples, and patient’s or volunteer’s blood. The method is simple; it gives results comparable to those obtained by other techniques that require elaborate instrumentations and time-consuming sample preparation procedure.The method showed good accuracy and precision suitable for quality assurance and could be recommended for bioequivalency and bioavailability studies as well as for validation of cleaning methodology prior to line clearance.The proposed method application could be extended to cover all available pharmaceutical preparations for each of the chosen drugs.



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*Source: 281929-2012-02-14.xml*