Development and validation of analytical methods for the determination of some drugs used for the treatment of colon diseases
A thesis presented by
Ahmed Adel Ibrahim Mohamed Othman B.Sc. Pharmacy and Biotechnology, Faculty of pharmacy, German University in Cairo 2011
Submitted for partial fulfillment for the degree of Master in Pharmaceutical Sciences
(Pharmaceutical Chemistry)
Under the Supervision of
Prof. Dr. Ramzia Ismail Ibrahim El-Bagary Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University Head of Pharmaceutical Chemistry Department, Faculty of Pharmacy-Future University
Prof. Dr. Mohamed Mahmoud El-Kerdawy Professor of Medicinal Pharmaceutical Chemistry Faculty of Pharmacy, Mansoura University
Dr. Ehab Farouk Hassan Elkady Assistant Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University
Faculty of Pharmacy Cairo University 2017
English Abstract
Development and validation of analytical methods for the determination of some drugs used for the treatment of colon diseases
Different methods for the determination of some drugs used in the treatment of colon diseases, in bulk and dosage forms have been introduced in this thesis. The cited drugs are (balsalazide disodium, chlordiazepoxide, mebeverine hydrochloride, mesalazine, olsalazine sodium, and otilonium bromide). The chromatographic part includes a method for simultaneous determination of three commonly prescribed drugs used for treatment of inflammatory bowel diseases and was appled in ternary mixtures and dosage forms. Also, it involved simultaneous determination of olsalazine sodium and its degradation product 5-aminosalicylic acid (mesalazine) and was applied in binary mixtures and dosage form. Finally in this part, an HPLC method was introduced for the simultaneous determination of otilonium bromide and its degradation product 4- aminobenzioc acid and was applied in dosage form. On the other hand, spectrophotometric part includes simultaneous spectrophotometric determination of mebeverine hydrochloride and chlordiazepoxide in bulk and pharmaceutical dosage form using three chemometric techniques, classical least square(CLS), principal component regression (PCR) and partial least squares (PLS). Also, it involved simultaneous determination spectrophotometric determination of otilonium bromide with its degradation products 4- aminobenzoic acid and salicylic acid using CLS, PCR and PLS chemometric techniques. Also, this part includes derivative spectrophotometric methods in which a validated spectrophotometric method was introduced for simultaneous determination of mebeverine hydrochloride and chlordiazepoxide in binary mixture and pharmaceutical preparations using second derivative method for determination of mebeverine hydrochloride and first derivative method for determination of chlordiazepoxide and other validated fourth derivative spectrophotometric method used for simultaneous determination otilonium bromide with its degradation products 4-aminobenzioc acid and salicylic acid. Statistical analysis was carried out for comparing results of the proposed methods with the reference methods exhibiting good values and revealing insignificant difference.
Keywords: balsalazide disodium, chlordiazepoxide, mebeverine hydrochloride, mesalazine, olsalazine sodium, otilonium bromide, HPLC, Chemometric techniques, derivative spectrophotometric methods, pharmaceutical preparations.
I-1- General introduction
Colon diseases include inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS) and colonic cancer. The chief types of inflammatory bowel disease are Crohn's disease (CD) and Ulcerative colitis (UC). Inflammatory bowel diseases fall into the class of autoimmune diseases, in which the body's own immune system attacks elements of the digestive system[1].The difference between CD and UC is the location and nature of the inflammatory changes. CD can affect any part of the gastrointestinal tract, from mouth to anus, although a majority of the cases start in the terminal ileum. UC, in contrast, is restricted to the colon and the rectum[2]. Microscopically, UC is restricted to the mucosa (epithelial lining of the gut), while CD affects the full thickness of the bowel wall ("transmural lesions"). Lastly, CD and UC present with extra-intestinal manifestations (such as liver problems, arthritis, skin manifestations and eye problems) in different proportions[2]. In spite of CD and UC being very different diseases, both may be present with any of the following symptoms: abdominal pain, vomiting, diarrhea, rectal bleeding, severe internal cramps/muscle spasms in the region of the pelvis and weight loss. Anemia is the most prevalent extraintestinal complication of inflammatory bowel disease [3, 4]. Associated complaints or diseases include arthritis, pyoderma gangrenosum, primary sclerosing cholangitis, and non-thyroidal illness syndrome [5]. Associations with deep vein thrombosis [6] and bronchiolitis obliterans organizing pneumonia have also been reported [7]. Diagnosis is generally by assessment of inflammatory markers in stool followed by colonoscopy with biopsy of pathological lesions.
Colon associated diseases such as nervous colon or, IBS which is a group of symptoms including abdominal pain and changes in the pattern of bowel movements without any evidence of underlying damage [8]. These symptoms occur over a long time, often years [9]. There are three types of IBS: a- IBS with Constipation (IBS-C): this comes with stomach pain and discomfort, bloating, abnormally delayed or infrequent bowel movement, or lumpy/hard stool. b- IBS with Diarrhea (IBS-D): this comes with stomach pain and discomfort, an urgent need to move your bowels, abnormally frequent bowel movements, or loose/watery stool. c- IBS with alternating constipation and diarrhea [10].
Disorders such as anxiety, major depression, and chronic fatigue syndrome, are common among people with IBS [8, 11]. The causes of IBS are not clear. Theories include gut–brain axis problems, small intestinal bacterial overgrowth, genetic factors, food sensitivity, and gut motility problems [9]. Onset may be triggered by an intestinal infection [12], or stressful life event [13]. IBS is a functional gastrointestinal disorder [8].
Ulcerative colitis (UC) is a chronic inflammatory disorder of colon. This inflammation may be limited to the left-hand side of the colon or extend to entire colon. Colon related pathologies range in seriousness from constipation and diarrhea to the incapacitating inflammatory bowel diseases through to colon cancer, the third most widespread form of cancer in both women and men. Carcinoma of the colon afflicts patients with ulcerative colitis 7–30 times more frequently than it does the general population. The risk of colon cancer in ulcerative colitis is related to two factors: (1) duration of the colitis, and (2) extent of colonic involvement. The colonic malignancy associated with ulcerative colitis is generally an adenocarcinoma evenly scattered throughout the colon [14].
Figure (1): Anatomic distribution of Crohn’s disease and Ulcerative colitis [15] .
1-Some important drug classes of colon diseases Drugs used for treatment of IBS a-Muscarinic antagonists One of the most important compounds in this class is hyoscine, it acts on the relaxation of the smooth muscles of the gut [16], it is used as antispasmodic to reduce the activity of the gastrointestinal tract. Dicyclomine and hyoscyamine are also antispasmodics in IBS [16]. These medications can be given as an oral formulation or a sublingual tablet, and be dosed on an as-needed or regular basis. Many patients benefit by taking the medication before meals. A number of these agents are presented in Table (1).
Mebeverine hydrochloride acts directly on the gut muscles at the cellular level to relax them [17]. This relieves painful muscle spasms of the gut without affecting its normal motility. Mebeverine possess a phenylethylamine moiety which affects the colonic motility as well as gastric motility and has been used in treatment of IBS [18]. Table (1).
Table (1): Most commonly used Antispasmodics.
Drug Structure Trade name
Dicyclomine HCl .HCl Spasmorest®
Hyoscine N- Buscopan® Butylbromide
Mebeverine Coloverin SR®
Otilonium bromide Spasmomen®
b-Calcium antagonists
Pinaverium Bromide belongs to this class which inhibits the calcium influx by blocking the voltage dependent calcium channels at the smooth muscle cell level. It is selective for the intestinal smooth muscle [19]. It is used to treat IBS by relieving the symptoms of abdominal pain, bowel disturbances and intestinal discomfort [20]. Structure activity relationship (SAR) study reveals that quaternary ammonium derivatives such as pinaverium bromide have been discovered and developed as potent spasmolytic of the gastrointestinal tract. Table (2).
Table (2): Most commonly used Calcium antagonists.
Drug Structure Trade name Pinaverium Bromide Spascolon®
c-Adjuvant therapy for IBS
Although many drugs are available to treat the symptoms of irritable bowel syndrome, these drugs do not cure the condition. They are primarily used to relieve symptoms. The choice among these medications depends in part upon whether the patients have diarrhea, anxiety or pain-predominant irritable bowel syndrome such as:
1-Anti-anxiety drugs Anti-anxiety drugs reduce anxiety. Chlordiazepoxide (Librax®), Diazepam (Valium®), lorazepam (Ativan®), and clonazepam (Klonopin®) belong to this class of drugs. Anti-anxiety drugs are occasionally prescribed for people with short-term anxiety that is worsening their irritable bowel syndrome symptoms [21]. Table (3).
Table (3): Most commonly used Anti-anxiety.
Drug Structure Trade name
Chlordiazepoxide Librax®
Diazepam Valium®
2-Tricyclic Antidepressants: Many tricyclic Antidepressants (TCAs) have a pain relieving effect in people with irritable bowel syndrome. Tricyclic Antidepressants in IBS patients can facilitate endogenous endorphin release, blockade of norepinephrine leading to enhancement of descending inhibitory pain pathways, and blockade of the pain neuromodulator, serotonin [22, 23]. Table (4).
Table (4): Most commonly used Tricyclic Antidepressants.
Drug Structure Trade name
Amitriptyline Tryptizol®
Imipramine Tofranil®
Other lines of conservative management of IBS symptoms:
Exercise and Fiber intake
Exercise can help to maintain gastrointestinal function and reduce stress and yoga practice can also help to relieve some IBS symptoms [24-27]. The addition of psyllium fiber, especially for persons with IBS-C, reduced IBS symptoms [28].
Drugs used for treatment of IBD
a-Glucocorticoids
Glucocorticoids are potent anti-inflammatory drugs. The drugs of choice are usually prednisolone or budesonide (although others can be used). They are administered orally or locally into the bowel by suppository or enema [16]. Table (5).
Table (5): Most commonly used Glucocorticoids.
Drug Structure Trade name Prednisolone Solupred®
b-Aminosalicylates
While glucocorticoids are useful for the acute attacks of inflammatory bowel diseases, they are not the ideal for the long-term treatment because of their side effects. Maintenance of remission in both UC and CD is generally achieved with aminosalicylates, although they are less useful in the latter condition [16]. Sulfasalazine consists of the sulfonamide sulfapyridine linked to 5- aminosalicylic acid (5-ASA). The latter forms the active moiety when it is released in the colon. Its mechanism of action is obscure. It may reduce inflammation by scavenging free radicals, by inhibiting prostaglandin and leukotriene production, and/or by decreasing neutrophil chemotaxis and superoxide generation [16]. Newer compounds in this class, which presumably share a similar mechanism of action, include mesalazine (5-ASA itself), olsalazine (a 5-ASA dimer linked by a bond that is hydrolysed by colonic bacteria) and balsalazide; a prodrug from which 5-ASA is also released following hydrolysis of a diazo linkage [16]. Mesalazine is comparable with newer substances (balsalazide) in its efficacy to induce remission and is better tolerated [29-31]. Table (6). Table (6): Most commonly used Aminosalicylates. Drug Structure Trade name Balsalazide Colozal®
Mesalazine Pentasa®
Olsalazine Na Dipentum®
c-Other Drugs:
- Thiopurines are purine antimetabolites which are widely used in the treatment of autoimmune disorders (e.g., CD, rheumatoid arthritis), and organ transplant recipients [32]. Azathioprine (AZA) was shown to be efficient in inducing and maintaining remission after tapering of steroids. It is used in the management of moderately to severely or chronically active CD and in corticosteroid dependent patients [33, 34]. - Methotrexate (MTX) is a folic acid inhibitor and thus interferes with cell growth [35]. It is used predominantly for immunosuppression in autoimmune diseases and as chemotherapy. In CD it has to date remained in treatment algorithms as a salvage therapy for patients who have failed to respond or became intolerant to azathioprine [36]. Table (7).
Table (7): Other drugs used for treatment of colon diseases:
Drug Structure Trade name
Azathioprine Imuran ®
Methotrexate Methotrexate ®
Drugs used for treatment Colon cancer.
The drugs most often used for colorectal cancer include: - Fluorouracil (5-FU), which is often given with the vitamin-like drug leucovorin (also called folinic acid) or a similar drug called levo-leucovorin, which helps it to work better. - Capecitabine (Xeloda), is a prodrug converted to 5-FU when it gets to the tumor site [14]. Table (8). Table (8): Most commonly used Antimetabolites
Drug Structure Trade name Utoral® Fluorouracil
Xeloda® Capecitabine
- Aim and basis of the present investigation.
The wide spred of drugs used for the treatment of colon disease in medicine promoted the development of simple, accurate and applicable methods for their determination in bulk, laboratory prepared mixtures and dosage forms. The aim of this work was to develop simple accurate, precise and sensitive methods for the determination of some drugs used for the treatment of colon diseases either alone or in combination with other drugs. The drugs were analyzed in their pure form and in their commercially used pharmaceutical dosage forms using analytical techniques such as spectroscopy and chromatography. Drugs cited in this thesis include antispasmodics (otilonium bromide, mebeverine hydrochloride,), Benzodiazepines (chlordiazepoxide) and ulcerative colitis drugs (olsalazine sodium, balsalazide disodium and mesalazine).
HPLC is a powerful analytical tool for the separation and analysis of pharmaceutical products. It is incorporated in the plan of this work using ultraviolet detection for the detection of otilonium bromide with it’s degradation product para aminobenzoic acid. Otilonium bromide undergoes hydrolysis to yield degradation products; p-[2-(n-octyoxy) benzoyl]-aminobenzoic acid, and further hydrolysis yields (4-aminobenzoic acid, salicylic acid and diethyl-2-hydroxyethyl-methyl ammonium bromide) [163,164]. Also otilonium bromide was determined in laboratory prepared mixtures and dosage form. Olsalazine sodium was determined with its degradation product 5-amino salicylic acid in laboratory prepared mixtures and dosage form. Balsalazide disodium with olsalazine sodium and mesalazine were estimated in their laboratory prepared mixtures and dosage form.
Derivative spectrophotometry was suggested for enhancing the resolution of overlapping bands displayed by otilonium bromide with its degradation products 4- aminobenzioc acid and salicylic acid in their ternary mixtures, and for mebeverine hydrochloride with chlordiazepoxide in their binary mixtures.
Chemometric techniques were suggested for resolving spectral overlap displayed by otilonium bromide with its degradation products 4-aminobenzioc acid and salicylic acid in their ternary mixtures and for mebeverine hydrochloride with chlordiazepoxide in their binary mixtures. The plan of the work comprises the utilization of the proposed methods for the determination of the studied drugs in pure form and in their pharmaceutical formulations in addition to validation of these methods. Statistical comparison between the results obtained by the proposed methods and that of reference methods was performed in addition to statistical comparison in between the results of the methods.
I-3- Literature Review
1-3-1- Balsalazide disodium a- Pharmacopeial methods [37].
U.S.P. Pharmacopoeia [37], described acid-base titration method for the determination of balsalazide disodium in pure form using 0.1 M hydrochloric acid as titrant, endpoint was determined potentiometrically and described an HPLC method for balsalzide determination in pharmaceutical dosage form using mixture of acetonitrile: potassium phosphate buffer (1:4, v/v), as mobile phase at a flow rate of 1ml/min, column temperature 30⁰ C and UV detection was carried out at 360nm.
b- Chromatographic methods
Chromatographic methods reported for the determination of balsalazide disodium are listed in table (9).
Table (9): Chromatographic methods for the determination of balsalazide disodium.
Stationary phase Mobile phase Detector Application Ref
Eurosphere – 100 Water: Acetonitrile UV detection Estimation of C-18 (250x 4 mm (45:55, v/v). at 268 nm. balsalazide in bulk stainless steel with and pharmaceutical [42] 5 μm particle dosage form. size)column.
CrestPack RP C18 Ammonium formate UV detection Estimation of (250 mm x 4.6 mm buffer (pH adjusted to 3.5 at 361 nm. balsalazide disodium I.D; 5 μm particle with diluted formic acid): dihydrate in bulk and [43] size) column. Acetonitrile: MeOH Its pharmaceutical (65:25:1, v/v/v). formulations.
ODS 3V C18 (250× KH2PO4:ACN:MeOH UV detection Development and 4.6 mmID, 5 μm) at 304 nm. validation of column. (50:30:20, v/v/v) pH 4.5. balsalazide in bulk [44] drug.
Zorbax Eclipse Simple mobile-phase UV detection Analysis of XDB C18(150 mm gradient prepared from at 240 nm. balsalazide disodium [45] x 4.6 mm,5m methanol and phosphate in the bulk drug and particle) column. buffer (10 mM potassium in pharmaceutical dihydrogen dosage forms. orthophosphate monohydrate, adjusted to pH 2.5.
Inertsil ODS 3V KH2PO4: ACN: MEOH UV detection Development and [46] C18 (250x4.6 (50:30:20, v/v/v) pH 4.5. at 304 nm. validation of mmID, 5m balsalazide in bulk particle size) and capsule dosage column. form.
LunaC18 (250x4.6 Methanol: acetonitrile: UV detection Simultaneous mmID, 5m water (55:30:15, v/v/v). at 239 nm. determination of [47] particle size) paracetamol and column. balsalazide disodium dihydrate in combined tablet dosage Form.
Phenomenax Luna Acetonitrile: Methanol: UV detection Estimation of C18 (150×4.6mm; Triethylamine buffer at 254 nm. balsalazide in pure 5μm) column. (40:30:30, v/v/v). and in [48] pharmaceutical dosage forms.
Densitometric Methods
Stationary phase Mobile phase Detector Application Ref
Precoated silica gel Chloroform: methanol: UV detection Estimation of aluminium plate triethylamine (3.5:2:0.4, at 361 nm. balsalazide by 60F254. v/v/v). HPTLC-densitometry [49] method in pharmaceutical formulation.
c- Spectrophotometric methods:
Spectrophotometric methods reported for the determination of balsalazide disodium are listed in table (10).
Table (10): Spectrophotometric methods for the determination of balsalazide disodium.
Solvent/Reagent Method Wavelength Application Ref
Solvent: Water. Based on oxidation of UV A comparative [50] balsalazide with Chloramine-T detection at spectrophotometric Reagent: Chloramine- and bromamine-T. 357nm kinetic study of T, and Bromamine-T oxidation of balsalazide.
Solvent: Distilled Based on the principle that UVdetection Estimation of water, 0.1 M balsalazide can exhibit two at 354 nm balsalazide in bulk hydrochloric acid and different chemical forms in and 460 nm and in [51] for acidic in 0.1 M sodium basic and acidic medium that pharmaceutical and alkaline hydroxide. differ in the absorption spectra medium dosage form. in basic and acidic medium. respectively.
Solvent: Double In method (A), balsalazide in UV Quantitative distilled water. water exhibited absorption detection for estimation of maximum at 260 nm in UV method (A) balsalazide in bulk [52] at 260 nm Reagent in method region. Method (B) is based on drug and (B): Nitrous acid. and for the formation of nitrosation method (B) pharmaceutical product resulting from the at 344 nm. formulation. reaction of balsalazide with nitrous acid.
Solvent: Double Method (A) based on the UV Analysis of distilled water for absorbance of balsalazide in detection for balsalazide in bulk method (A), and water and measure the method (A) drug and in at358 nm, alkaline medium at absorbance at 358 nm. Method pharmaceutical for method [53] pH 12for method (B). (B) based on the formation of a (B) at 456 formulation using dark red colored chromogen in nm, and for three methods. Reagent: In method alkaline medium at pH 12. method (C) (B): alkaline medium Method (C) based on using first at 383 nm. at pH 12. derivative spectroscopic method.
Solvent: Double Method (A): based on the UV Quantitative distilled water. oxidation followed by complex detection for estimation of formation reaction of method (A) balsalazide in bulk at 509 nm, Reagents: balsalazide with 1, 10- drug and dosage and for [54] Method (A): 1, 10- phenanthroline in presence of method (B) form. phenanthroline in ferric chloride to form blood at 790 nm. presence of ferric red colored chromogen. chloride. Method (B): based on the oxidation followed by complex Method(B):potassium formation reaction of ferricyanide in balsalazide with potassium presence of ferric ferricyanide in presence of chloride. ferric chloride to form a bluish green colored chromogen.
1-3-2- Chlordiazepoxide a- Pharmacopeial methods [37, 40].
In the U.S.P. Pharmacopoeia [37] and B.P. [40] reported a non-aqueous titration method for determination of chlordiazepoxide in pure form using 0.1M perchloric acid as titrant , the endpoint was determined potentiometrically, while U.S.P. Pharmacopoeia [37] reported an HPLC method for determination of chlordiazepoxide in pharmaceutical dosage form, using mixture of methanol: water (60:40, v/v), as mobile phase at a flow rate of 1ml/min and UV detection was carried out at 254nm.
b- Chromatographic methods:
Chromatographic methods reported for the determination of chlordiazepoxide are listed in table (11).
Table (11): Chromatographic methods for the determination of chlordiazepoxide.
Stationary Mobile phase Detector Application Ref phase ODS-3 column Mixture of 0.05 mol / L UV Determination of seven (125 x 4 mm ammonium dihydrogen detection at frequently prescribed 1,4- [55] i.d., 5 μm phosphate solution : 254 nm. benzodiazepines in bulk particle size). methanol (50:50, v/v) which powder or formulated in was adjusted to pH 5.8. tablets or capsules. Phenomenex The following mobile phases UV Simultaneous analysis of RP C18 were used detection at four benzodiazepines in (250 mm x 4.6 A Methanol: Water 254 nm. pharmaceutical formulations. [56] mm, 5μ) (60:40). column. B Methanol: Acetonitrile: Water (45:40:15). C Methanol: Acetonitrile: Water (70:25:5). D Methanol: Acetonitrile (60:40).
Bondapak'" Mobile phase consisted of UV Micro-extraction of C18-Corasil deionized water/ detection at chlordiazepoxide and its column methanol/acetonitrile/acetic 240 nm. primary metabolites, [57] (300 mm x 3.9 acid (51:28:16:0.2, desmethylchlordiazepoxide mm, 5μm). v/v/v/v; pH 3.7). and demoxepam, from plasma. Kromasil C18 Using a mobile phase ratio UV Simultaneous determination (250 mm × 4.6 consisting of (40:30:30) detection at of clidinium bromide, mm id, 5μm) Methanol: Acetonitrile: 270 nm. chlordiazepoxide and [58] column. Potassium dihydrogen dicyclomine hydrochloride in phosphate buffer (0.05M, pH bulk and combined tablet 4.0). dosage forms. Phenomenex Potassium dihydrogen UV Simultaneous determination Luna C18, phosphate buffer (0.05 M, pH detection at of clidinium bromide and [59] (250 mm x 4.6 4.0 adjusted with 0.5% 220 nm. chlordiazepoxide in mm, i.d., 5m) orthophosphoric acid)– combined dosage forms. column. methanol–acetonitrile (40:40:20, v/v/v). LC- 2010 AT Buffer (pH 3.5): Acetonitrile: UV Simultaneous estimation of C18, TEA (80:20:0.1, v/v/v) as detection at trifluoperazine, (250mm x 4.6 mobile phase. 228 nm. chlordiazepoxide and mm x 5 μm) trihexiphenidyl in its [60] column. pharmaceutical dosage form.
Agilent Zorbin Potassium dihydrogen UV Simultaneous estimation of [61] C18, (250 X phosphate buffer: Acetonitrile detection at amitriptyline HCl and 4.6 mm, 5μm) in the ratio of (50:50, v/v). 274 nm. chlordiazepoxide. column. Nucleodur C8, CH3CN-MeOH-NH4OAc UV Simultaneous determination (250x4.6 mm 0.1M (30: 40: 30, v/v/v) was detection at of clidinium bromide [62] i.d., 5 μm used as the mobile phase. 218 nm. and chlordiazepoxide in particle size) combined dosage forms. column. Phenomenex Methanol: water- (97:03, v/v). UV Estimation of C18 (250×4.6 detection at Trifluoperazine mm id, 5 μm 262 nm. hydrochloride and [63] particle chlordiazepoxide in tablet size)column. dosage forms. Zodiac C18 Methanol: acetonitrile (75:25, UV Validation of (250mm x v/v). detection at chlordiazepoxide and [64] 4.6mm x 5μm), 221 nm. amitryptyline hydrochloride column. in pharmaceutical formulation. Phenomenex phosphate buffer(pH3) : UV Simultaneous estimation of C18(150x4.6m Acetonitrile( 55:54, v/v). detection at amitryptyline and [65] m,5μm) 252 nm. chlordiazepoxide. column. C18 (250×4.6 0.1ml Tri ethyl amine in UV Simultaneous estimation of mm, 5 μm 0.01M KH2PO4 buffer pH3.5: detection at chlordiazepoxide, particle size) acetonitrile (80:20, v/v). 247 nm. trifluoperazine hydrochloride column. and trihexyphenidyl [66] hydrochloride in tablet dosage form. Waters C18 Methanol: Acetonitrile: 0.2M UV Simultaneous estimation of reverse‐phase Orthophosphoric acid, pH4.59 detection at amitriptyline hydrochloride (250 mm × 4.6 (56:24:20, v/v/v). 240 nm. and chlordiazepoxide [67] mm, 5μm) in tablet dosage form. column. Zodiac C-18 Methanol: Acetonitrile (70:30, UV Determination of column (250 X V/V), the pH was adjusted to detection at amitriptyline and 4.6 mm, 5μm). 4.9 with Triethyl amine. 222 nm. chlordiazepoxide in [68] combined pharmaceutical dosage form. Chromosil C- Methanol: UV Determination of 18 Acetonitrile: 0.1%OPA in detection at chlordiazepoxide in (250x4.6mm, (50:40:10, v/v) pH 4.7. 217 nm. formulations. 5μm in particle [69] size) column. UPLC HSS T3 Solvents A (2 mmol/L MS/MS. Quantification of 29 drugs of C18 column, ammonium acetate, pH 6.2) abuse in oral fluid. [70] (100 mm × 2.1 and B (100% methanol). By mm, 1.8 μm). gradient programs. Xterra Methanol/formic acid MS/MS. Quantitative analysis of 33 MS C-18 approximately 0.006M benzodiazepines, metabolites (150mm × 2.1 ( 30:60, v/v). and benzodiazepine-like mm, 3.5m PS, substances in whole blood by [71] Waters) and the liquid chromatography– Xterra RP C-18 (tandem) mass spectrometry. (150mm × 2.1 mm, 3.5m PS, Waters). Acquity C18 Gradient elution of10 mM MS/MS. Evaluation of the column (1.7 ammonium bicarbonate in applicability of LC tandem m particle water set at pH 9.0 (solvent mass spectrometry analysis [72] size, 2.1 mm A) and methanol (solventB). of benzodiazepines and × benzodiazepine-like 50 mm). hypnotics in urine and blood. Hypersil The mobile phase consisted of MS/MS. Screening and quantification GOLD formic acid method of 87 psychoactive column (100 0.1 % (A) and methanol (B). drugs and their metabolites [73] mm, 2.1 mm Chromatographic gradient in hair. i.d., 3 m Elution. particle size).
c- Spectrophotometric methods: Spectrophotometric methods reported for the determination of chlordiazepoxide are listed in table (12).
Table (12): Spectrophotometric methods for determination of chlordiazepoxide.
Solvent/Reagent Method Wavelength Application Ref Solvent: Amitriptyline HCl and UV detection for Simultaneous estimation Methanol. chlordiazepoxide showed amitriptyline at of amitriptyline HCl and [74] maxmimum absorbance in 222 nm, and for chlordiazepoxide in methanol at 222nm and chlordiazepoxide combined pharmaceutical 251nm. The method was at 251 nm. dosage form. validated as per the ICH guidelines. Solvent: Method I: based on using UV detection for Determination of 0.1M HCl. multicomponent analysis. chlordiazepoxide chlordiazepoxide and Method II: based on using at 245 nm, and for trifluoperazine HCl from simultaneous equations trifluoperazine combined dosage form. [75] were applied for HCl at 255.5 nm. determination of both drugs. Solvent: Based on using the zero- UV detection for Determination of Acetonitrile. crossing method for chlordiazepoxide chlordiazepoxide and clidinium bromide and at283.6 nm, and clidinium bromide in [76] graphical method for for clidinium pharmaceutical chlordiazepoxide. bromide at 220.8 formulations by derivative nm. spectrophotometry. Solvent: Method 1 based on UV detection for Simultaneous estimation Methanol. involved determination of method I and II of amipramine HCl and imipramine HCl and for imipramine chlordiazepoxide in chlordiazepoxide using the HCl at 251 nm, tablets. simultaneous equations. and for [77] Method II based on using chlordiazepoxide the absorbance ratio at 264.5 nm. For method. Method III based method III first- on using the 1st derivative derivative spectrophotometric technique at 219 technique. nm and 231.5 nm for amipramine HCl and chlordiazepoxide. Solvent: The method is based on UV detection for A rapid method is 0.1N H2SO4 measuring the maximum the second described for the absorbance difference derivative of quantitative [78] existing between a chlordiazepoxide determination of maximum and minimum, is measured at benzodiazepines, by contiguous to the spectra 270 nm and 254 means of corresponding to their nm. derivative spectroscopy., second derivatives. Solvent: Chlordiazepoxide UV detection at Estimation of Distilled water. extracted from blood and 550 nm. Chlordiazepoxide in blood hydrolysed to 2- amino-5- and brain rats. [79] chloro-benzophenone and coupled with N-(1- naphthyl)ethylene diamine.
1-3-3- Mebeverine Hydrochloride
a- Pharmacopeial methods [40].
In the B.P. [40], mebeverine hydrochloride in bulk was determined by non-aqueous titration method using 0.1 M perchloric acid as titrant, the endpoint was determined potentiometrically, while B.P. [40] reported UV spectrophotometric method for determination of mebeverine in pharmaceutical dosage form by measuring the absorbance of mebeverine hydrochloride solution at 263nm using 0.1M hydrochloric acid as solvent. b- Chromatographic methods: Chromatographic methods reported for the determination of mebeverine hydrochloride are listed in table (13).
Table (13): Chromatographic methods for the determination of mebeverine hydrochloride.
Stationary phase Mobile phase Detector Application Ref
Bondapak 125 a ACN:H2O (1:1, v/v). UV detection Determination of mebeverine C18 column (250 at 205 nm. hydrochloride in raw materials [80] mm 4.6 mm, and tablets. particle size 10 μm). Lichroma RP8 MeOH:H2O (75:25) UV detection Determination of mebeverine column (15 cm, containing 0.05% at 220 nm. hydrochloride in tablets. 4.0 i.d., 5 μm). hexylamine, adjusted [81] pH to 5. Bondapack C18 0.05 M ammonium UV detection Quantitative analysis of [82] column(3.9x30cm acetate buffer and at 263 nm. mebeverine in dosage forms. ,10 μm).3 3.9 x acetonitrile, [(45%, 300 mm.9 x 300 v/v) pH 5.2]. mm Symmetry C18 Mixture of 50 mM UV detection Kinetic study of degradation of [83] column (15 cm, KH2PO4, acetonitrile at 263 nm. mebeverine in oxidative 4.0 i.d., 5 μm). and THF (63:35:2, condition. v/v/v). Symmetry C18 Mixture of 50 mM UV detection Mebeverine hydrochloride in the column (4.6 mm KH2PO4, acetonitrile at 263 nm. presence of its degradation [84] × 150 mm, 5 μm, and tetrahydrofuran products. Waters). (THF) (63:35:2; v/v/v). U-Bondapack C18 Water: Acetonitrile: Excitation Determination of mebeverine column Acetic acid (59:40:1) and emission hydrochlride in plasma using (3.9mmx30cm, 10 mixture. wave lengths fluorescence detection. [85] µm). 270 and 362 nm, respectively. Chiralcel OD n-Hexane, isopropyl UV detection Determination of mebeverine column. (250 mm alcohol and at 263 nm. hydrochlride in pharmaceutical [86] × 4.6 i.d., 10 μm triethylamine dosage forms and spiked rat particle (90:9.9:0.1, v/v/v). plasma. size). DELTA S column Hexane-isopropyl UV detection Application to pharmacokinetics (250mmx4.6mm alcohol (85:15, v/v) at 233 nm. study in rat plasma. [87] i.d., 5 m particle containing 0.2% size). triethylamine. Lichroma Methanol-water UV detection Determination of mebeverine column(( (250 x mixture (75-25, v/v), at 220 nm. hydrochlride in tablets. 4.6mm i.d., containing 0.05% [88] packed with hexylamine as silanol- 10m).diameter blocking agent. Phenomenex&® Methanol: acetonitrile: UV detection Determination of mebeverine Luna C18 water (45:40:15, at 254 nm. hydrochloride and [89] (250mm x 4.6mm v/v/v). chlordiazepoxide in commercial i.d., 5µm ps). tablets. analytical column. RP-C8 (octylsilyl) Acetonitrile-0.05 M UV detection Determination of mebeverine analytical column disodium hydrogen at 247 nm. hydrochloride in tablets. [90] (250 mm × 4.6 phosphate- mm, 5 μm particle triethylamine (50:50: size). 0.2, v/v/v). Agilent C18 Methanol : Tri ethyl UV detection Determination of mebeverine and column. (250 amine (40:60, v/v) at 262 nm. chlordiazepoxide In [91] mm×4.6 mm i.d., buffer pH 7.0 with pharmaceutical preparations. 5 μm particle OrthoPhosphoric size). Acid. RP cyano column. Acetonitrile: water UV detection Determination of sulpiride and (5m, 250 mm x (70:30, v/v) adjusted to at 221 nm. mebeverine hydrochloride in [92] 4.6 id). pH = 7. combination.
® Waters -C18 Acetonitrile and 0.01 Fluorescence Determination of sulpiride and column. (250 mm, M -detection at mebeverine to real human 4.6 mm i.d., 5 m dihydrogenphosphate excitation particle size). buffer (45:55) at pH = 300 nm and plasma. [93] 4.0. emission at 365 nm. X-Bridge C18 Gradient elution UV detection Development and application of a (150 mm × 4.6 system. at 230 nm. novel, dual-mode gradient, [94] mm i.d., 3.5 μm stability-indicating method for particle size). the simultaneous determination and purity assessment of mebeverine hydrochloride, diloxanide furoate and their corresponding major degradation products. RP C18 column. Gradient elution UV detection Development of gradient HPLC- [95] (150 mm × 4.6 system. at 260 nm. DAD method for assay of ternary mm, 5 μm). mixture containing amebicide and analgesic drugs. BDS Hypersil Acetonitrile: 0.1M UV detection Chlordiazepoxide and phenyl column potassium dihydrogen at 260 nm. mebeverine HCl in the presence (4.5 mm × 250 m phosphate: of their degradation products and m, 5 µm particle triethylamine impurities. [96] size). (35:65:0.2, v/v/v).
ODS column Water: methanol: UV detection Analysis of some amoebicide [97] (150×4.6 mm triethylamine (25: 75: at 230 nm. drugs in their combined I.D., 5 µm particle 0.5, v/v/v). pharmaceutical preparation. size). Alltima C18 (50 MeOH : 2mM MS/MS. Simultaneous quantification of [98] x4.6 mm), 5μm Ammonium formate mebeverine and its metabolits by particle size. (90:10, v/v). LC-MS/MS. Densitometric methods
Aluminum plates Diethyl ether: UV detection Mebeverine hydrochloride and [99] precoated with Methanol: Ethyl at 273 nm. chlordiazepoxide in their silica gel 60F254. acetate: Triethylamine combined pharmaceutical. (7:2:1:0.1, v/v/v/v). dosage form. HPTLC F 254 Ethanol: methylene UV detection Determination of sulpiride and plates. chloride: triethyl at 221nm. mebeverine hydrochloride in [92] amine (7:3:0.2, v/v/v). combination.
Silica gel GF254 Ethanol: diethyl ether: 262nm and Determination of sulpiride and plates. triethylamine (70:30:1, 240 for mebeverine hydrochloride. v/v/v). mebeverine [100] hydrochloride and sulpiride respectively. Pre-coated plates Acetone: methanol: UV detection Determination of alprazolam [101] with silica gel 60F acetic acid (6:4:0.1, at 225 nm. and mebeverine, both as bulk 254. v/v/v). drugs and in formulations. Pre-activated Hexane: acetone: UV detection Determination of [102] silica gel 60F254 triethyl amine (7: 3: at 254 nm. metronidazole, diloxanide TLC plates. 0.6, v/v/v). furoate, and mebeverine hydrochloride in tablets. c- Spectroscopic methods:
Spectroscopic methods reported for the determination of mebeverine hydrochloride are listed in table (14).
Table (14): Spectroscopic methods for determination of mebeverine hydrochloride.
Solvent/Reagent Method Wavelength Application Ref Detection Solvent: Based on the formation UV detection with Determination of Chloroform. of chloroform soluble reagent (1) at 416 nm mebeverine Reagents: ion-pair complexes of and with reagent (2) hydrochloride in pure 1- Bromophenol mebeverine with at 524 nm. and pharmaceutical [103] blue. bromophenol blue,and formulations. 2- Erichrome with Erichrome black to Black. form yellow and pink colored resp. Solvent: Three methods were UV detection with Determination of Deionized water. used: reagent (A) at 625 mebeverine Reagents: A and B,based on the nm, with reagent (B) hydrochloride in bulk A- Fast green. B- formation of ion- at 405nm, and with samples and Bromothymol blue. association complexes reagent (C) at 625 pharmaceutical C- Cobalt between the drug and nm. formulations. [104] thiocyanate. fast green for method(A) ,and bromothymol blue for method (B) and method (C) based on the formation complex with cobalt thiocyanate. Solvent: Deionized Two methods were UV detection for Determination of water. developed for method (1) at 551nm. mebeverine HCl in Reagent: determination Method II: The tablets by eosin Y. mebeverine HCl: native fluorescence of spectrophotometric and [105] Method I: Based on a eosin Y at 540 nm spectrofluorimetric binary complex after excitation at 390 methods. formation with eosin Y. nm. Method II: Based on spectrofluorimetric method depend on measuring the quenching effect of the drug on native fluorescence of eosin Y. Solvent: Based on determination UV detection at Evaluation of Deionized of mebeverine 246 nm. mebeverine water/ethanol hydrochloride at zero hydrochloride in bulk. [106] (96%). order. Solvent: Based on determination UV detection at Quantitative Deionized water. of mebeverine by wavelength’s 258, determination of multivariate calibration 260, 2622, 264, and mebeverine in bulk drug approach. The approach 266 nm. and pharmaceutical [107] bases on the use of formulations. linear regression analysis equation by using correlation of concentration of drugs and corresponding absorbance taken at five unlike wavelengths. Solvent: Based on the assay of UV detection at Determination of Triethylammonium mebeverine 260 nm. mebeverine phosphate buffer hydrochloride and hydrochloride and [108] (pH=3). chlordiazepoxide in chlordiazepoxide in capsules. MEVA C capsules. Solvent: Based on the assay of UV detection at Analysis of mebeverine Triethylammonium mebeverine 263 nm and 245 nm, hydrochloride and phosphate buffer hydrochloride and for mebeverine and chlordiazepoxide in [109] (pH 4.0). chlordiazepoxide in chlordiazepoxide MEVA C capsules. capsules. respectively. Solvent : Method I: Based on UV detection at 220 Quantitative estimation Methanol applying the nm and 264.5nm for of mebeverine simultaneous equations. mebeverine and hydrochloride and Method II; Based on chlordiazepoxide chlordiazepoxide in [110] applying Q-analysis respectively. tablet dosage form. (absorbance ratio method). Solvent: Based on comparison UV detection for Analysis of mebeverine Absolute ethanol. between support vector mebeverine at hydrochloride and regression and Artificial 221nm, and for sulpiride mixtures in Neural Networks for sulpiride at 232 nm. pharmaceutical [111] quantitative analysis of preparation. mixtures of mebeverine and sulpiride. Solvent: Based on first-derivative UV detection for the Determination of [99] Methanol. ultraviolet first derivative sulpiride and spectrophotometry, with amplitudes at 214.2 mebeverine zero-crossing and 221.6 nm were hydrochloride in their measurement method. selected for the assay combined tablets. of mebeverine and sulpiride. Solvent: Two methods are UV detection of the Determination of 0.1M HCl for described for the first method at 263.7 mebeverine chemometric simultaneous nm and 234.9 nm for hydrochloride and method. determination of mebeverine and sulpiride in the mebeverine sulpiride, pharmaceutical [112] hydrochloride and respectively. formulation of Colona sulpiride in their tablets. combinations. The first method depends on the first derivative of the ratio spectra. The second method based on chemometric (classical least squares) method. Solvent: Based on the analysis of UV detection of Analysis of mebeverine Methanol. mebeverine alprazolam and hydrochloride and hydrochloride and mebeverine by alprazolam in bulk drugs alprazolam by three method (A), at and pharmaceutical [113] UV methods. Method 220.45 nm and dosage form. (A) by 1st derivative. 242.55 nm. Method Method (B) by (B), difference in difference between two absorbance between absorbances 217 and 227nm, and wavelength’s, and difference between method (C) by 257nm and 267nm simultaneous equation for dual wavelength. method. Method (C) at 222nm and 262.40nm.
Solvent: Based on two methods : The mean centering Determination of some Methanol. (1) the mean centering ratio spectra at 293- antiprotozoal drugs in of ratio spectra method, 311 nm (peak to peak different combined and method (2): the for mebeverine. dosage forms and in partial least square dimetrol suspension. (PLS) multivariate [114] calibration method. Solvent: Based on determination UV detection of Analysis of some Methanol. of mebeverine HCl and mebeverine HCl at amoebicide drugs in diloxanide furoate using 226.4 nm-232.2 nm their combined [97] derivative ratio spectra (peak to peak), pharmaceutical method, which depends diloxanide furoate at preparation. on derivativatization of 260.6 nm-264.8 nm the obtained ratio (peak to peak), and spectra in two steps. metronidazole at 327 While metronidazole by nm using first first derivative. derivative. Solvent: Based on reaction of the UV detection at Determination of Dichloromethan. selected drugs with 422 nm. benzydamine HCl, methyl orange (MO) in levamisole HCl and Reagent: buffered aqueous mebeverine HCl through [115] Methyl orange. solution at pH 3.6. ion-pair complex formation with methyl orange. Solvent: Based on application of First derivative of Application of fourier Methanol. double divisor ratio these ratio spectra function to double spectra derivative were obtained at divisor ratio spectra method and hybrid and the peak curves for analysis of double divisor ratio amplitudes at some amoebicide drugs spectra method for 229.2nm were plotted (mebeverine [116] determination of against the hydrochloride, mebeverine corresponding diloxanide furoate and hydrochloride in ternary mebeverine metronidazole) in their mixtures. concentration. ternary mixtures. . Solvent: Based on: 1-UV detection of Colorimetric 1-Chloroform. 1- The reaction of mebeverine with determination of 2-and 3- mebeverine with iodine iodine at 229nm, 2- mebeverine acetonitrile. to give a yellow mebeverine with hydrochloride in tablets Reagent: molecular charge TCNE at 416nm, and by charge-transfer [117] 1- Iodine. transfer complex. 3-mebeverine complexation. 2- 2-&3- Formation of withTCNQ at 840nm. Tetracyanoethylene radical anions between (TCNE). mebeverine and TCNE 3- 7,7,8,8- or TCNQ. tetracyanoqui- nodimethane (TCNQ).
Spectrofluorimetric methods Solvent: Based upon measurement of The Analysis of mixtures of Water. the synchronous fluorescence fluorescence mebeverine intensity of these mebeverine intensities of the first hydrochloride and hydrochloride and sulpiride at derivative spectra sulpiride by using first [118] ∆λ = 100 nm in water. were derivative synchronous estimated at 238 and fluorometric method in 264 nm for tablets and in human mebeverine and plasma. sulpiride, respectively. Solvent For Two methods are described The second method is Determination of certain second for the quantitative based on measuring antispasmodic drugs method: determination of mebeverine the native mebeverine 0.1 N hydrochloride as single fluorescence of hydrochloride, and in sulphuric acid. ingredient. The first method mebeverine two component [119] depends on the application of hydrochloride at mixtures, mebeverine quantitative 1H-NMR 360 nm with hydrochloride-sulpiride spectroscopy. The second excitation at 290 nm. and isopropamide method is based on iodide-trifluoperazine measuring the native hydrochloride. fluorescence of mebeverine hydrochloride at 360 nm with excitation at 290 nm.