Arraying prostate specific antigen PSA and Fab anti-PSA using light-assisted molecular immobilization technology

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti‐PSA biosensor arrays using UV light‐assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti‐PSA in solution and immobilized Fab anti‐PSA cross‐reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.     

Age-specific reference ranges for prostate-specific antigen among healthy Syrian men

Prostate-specific antigen (PSA) has become the most useful serum tumor marker in adjunct to digital rectal examination (DRE) for the management of prostate cancer (PC). The currently cited reference range of normal PSA levels (between 0 and 4.0 ng/mL) is deficient in terms of both sensitivity and specificity. Age-adjusted PSA have been proposed as an alternative to serum PSA. The primary objective of the present study is to determine the age-specific PSA and the percentage of free PSA (%FPSA) in healthy Syrian men. A total of 3,000 healthy Syrian men over 40 years old participated in this study. All men were asked to have total serum (TPSA) and free PSA (FPSA) tested, a DRE, and, when recommended, a prostate biopsy. Serum TPSA levels correlated with age (r=0.30, p<0.001). The 95th percentile TPSA values ranged from 1.7 ng/mL in the age group 40-49 years to 5.8 ng/mL in the group 70-80 years. The general pattern for medians and the percentile values for FPSA across age groups were similar to those seen for TPSA concentrations (r=0.37, p<0.001). However, the %FPSA was fairly constant across age groups except for the higher ratios in the youngest age group. These findings confirm that the serum TPSA and FPSA levels correlate with age. Interestingly, however, Syrian men have lower PSA values compared with other races. Racial differences of PSA concentrations were observed. Our results may help increasing the sensitivity and specificity of PSA measurements in the diagnosis of prostate cancer in Syria and probably in the surrounding regions.     

Serum level of prostate-specific antigen (PSA) in women with breast cancer

ObjectiveTo identify the diagnostic role of total and free prostate-specific antigen (TPSA and FPSA) in breast cancer in women.MethodsBlood samples of 55 women with breast cancer were prospectively analyzed for PSA before and after breast surgery, with a control group of 82 healthy women.ResultsTotal and free PSA levels were significantly higher in women with breast cancer (preoperatively) than in healthy women (P < 0.001). Both serum TPSA and FPSA showed a significant decline in their pre-surgical values after surgical removal of the tumor (P < 0.001). A significant proportion of breast cancer patients (83.6%) had free PSA as the predominant molecular form in serum as compared to 0% of controls and 1.8% of postoperative groups (P < 0.001). TPSA and FPSA levels were significantly associated with younger age and earlier cancer stage, whereas no significant association was found between these two variables and FPSA as a predominant molecular form.ConclusionsThis study indicated a clinical significance of preoperative measurement of serum TPSA and FPSA in the diagnosis of women with breast cancer, and may be a useful marker for monitoring the response to treatment.     

An electrochemical biosensor for prostate cancer biomarker detection using graphene oxide–gold nanostructures

In this paper, a most sensitive electrochemical biosensor for detection of prostate‐specific antigen (PSA) was designed. To reach the goal, a sandwich type electrode composed of reduced graphene oxide/ gold nanoparticles (GO/AuNPs), Anti‐Total PSA monoclonal antibody, and anti‐Free PSA antibody was assembled. The functionalized materials were thoroughly characterized by atomic force microscope spectroscopy, transmission electron microscopy, and X‐ray diffraction techniques. The electrochemical properties of each of the modification step were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The results presented that the proposed biosensor possesses high sensitivity toward total and free PSA. Furthermore, the fabricated biosensor revealed an excellent selectivity for PSA in comparison to the other tumor markers such as BHCG, Alb, CEA, CA125, and CA19‐9. The limit of detection for the proposed electrochemical biosensor was estimated to be around 0.2 and 0.07 ng/mL for total and free PSA antigen, respectively.     

Comprehensive role of prostate‐specific antigen identified with proteomic analysis in prostate cancer

Current treatments including androgen deprivation fail to prevent prostate cancer (PrCa) from progressing to castration‐resistant PrCa (CRPC). Accumulating evidence highlights the relevance of prostate‐specific antigen (PSA) in the development and progression of PrCa. The underlying mechanism whereby PSA functions in PrCa, however, has yet been elucidated. We demonstrated that PSA knockdown attenuated tumorigenesis and metastasis of PrCa C4‐2 cells in vitro and in vivo, whereas promoted the apoptosis in vitro. To illuminate the comprehensive role of PSA in PrCa, we performed an isobaric tag for relative and absolute quantitation (iTRAQ)‐based proteomic analysis to explore the proteomic change induced by PSA knockdown. Among 121 differentially expressed proteins, 67 proteins were up‐regulated, while 54 proteins down‐regulated. Bioinformatics analysis was used to explore the mechanism through which PSA exerts influence on PrCa. Protein‐protein interaction analysis showed that PSA may mediate POTEF, EPHA3, RAD51C, HPGD and MCM4 to promote the initiation and progression of PrCa. We confirmed that PSA knockdown induced the up‐regulation of MCM4 and RAD51C, while it down‐regulated POTEF and EPHA3; meanwhile, MCM4 was higher in PrCa para‐cancerous tissue than in cancerous tissue, suggesting that PSA may facilitate the tumorigenesis by mediating MCM4. Our findings suggest that PSA plays a comprehensive role in the development and progression of PrCa.     

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe₃O₄@SiO₂ magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe₃O₄@SiO₂ and carboxylic acid‐functionalized Fe₃O₄@SiO₂). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC₅₀) values of the two methods were about 0.024 ng ml^?1 and 0.046 ng ml^?1 respectively and LODs were approximately 0.0002 ng ml^?1 and 0.001 ng ml^?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs.     

Effect of biolinker on the detection of prostate specific antigen in an interferometry

Prostate-specific antigen (PSA) has been identified as a significant biomarker for prostate cancer screening. Heavily-doped porous silicon, etched to form a Fabry-Perot fringe pattern, can be applied to an interferometric sensing for detecting PSA bound with PSA-antibody. In the previous works, a calyx crown derivative (Prolinker-A) was used as an alternative biolinker on the porous silicon surface for interferometric biosensing of DNA-damaging chemical instead of employing the conventional biomolecular affinity method using biotin, which resulted in a denser linker formation. In this study, G5 amineterminated PAMAM (poly amidoamine) dendrimer as a biolinker, was applied to an interferometric sensing of PSA by using porous silicon, which shows the enhancement of adhesion capability and increase of functional groups more than those with Prolinker-A. Considerably low level down to 1 ng/mL of PSA could be detected by this sensing system.     

Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling.

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.     

Flow‐injection analysis for meloxicam based on tris(2,2′‐bipyridine) ruthenium(II)–Ce(IV) chemiluminescent system

It was found that meloxicam could enhance the chemiluminescence (CL) of the tris(2,2'‐bipyridine) ruthenium(II)–Ce(IV) system in the medium of sulfate acid. Based on this phenomenon a new flow‐injection system with chemiluminescent detection has been proposed for determination of meloxicam. Under optimum conditions, meloxicam had a good linear relationship with the CL intensity in the concentration range of 6.0  10^?4 to 1.0 µg/mL and the detection limit was 3.7 × 10^?4 µg/mL. The proposed method was applied to detect meloxicam in tablets and a satisfactory recovery was obtained. The possible mechanism for this CL system is also discussed in this paper. Copyright © 2009 John Wiley & Sons, Ltd.     

Measurement of aberrant glycosylation of prostate specific antigen can improve specificity in early detection of prostate cancer

Introduction

We previously identified prostate cancer (PCa)-associated aberrant glycosylation of PSA, where α2,3-linked sialylation is an additional terminal N-glycan on free PSA (S2,3PSA). We then developed a new assay system measuring S2,3PSA using a magnetic microbead-based immunoassay. We compared the diagnostic accuracy of conventional PSA and percent-free PSA (%fPSA) tests.

Methods

We used MagPlex beads to measure serum S2,3PSA levels using anti-human fPSA monoclonal antibody (8A6) for capture and anti-α2,3-linked sialic acid monoclonal antibody (HYB4) for detection. We determined the cutoff values in a training test and measured serum S2,3PSA levels in 314 patients who underwent biopsy, including 138 PCa and 176 non-PCa patients with PSA of <10.0 ng/ml. Serum S2,3PSA levels were presented as mean fluorescence intensity (MFI). Receiver operating characteristic curves were used to evaluate the diagnostic accuracy of total PSA, %fPSA, and S2,3PSA.

Results

We determined an MFI cutoff value of 1130 with a sensitivity of 95.0% and specificity of 72.0% for the diagnosis of PCa in the training test. In the validation study, the area under the curve for the detection of PCa with S2,3PSA was 0.84, which was significantly higher than that with PSA or %fPSA.

Conclusions

Although the present study is small and preliminary, these results suggest that the measurement of serum S2,3PSA using a magnetic microbead-based immunoassay may improve the accuracy of early detection of PCa and reduce unnecessary prostate biopsy.     

A novel electroconductive interface based on Fe3O4 magnetic nanoparticle and cysteamine functionalized AuNPs: Preparation and application as signal amplification element to minoring of antigen‐antibody immunocomplex and biosensing of prostate cancer

In this study, a novel electroconductive interface was prepared based on Fe₃O₄ magnetic nanoparticle and cysteamine functionalized gold nanoparticle. The engineered interface was used as signal amplification substrate in the electrochemical analysis of antibody‐antigen binding. For this purpose, biotinilated‐anti‐prostate‐specific antigen (PSA) antibody was bioconjugated with iron oxide magnetic nanoparticles (Fe₃O₄) and drop‐casted on the surface of glassy carbon electrode (GCE). Also, secondary antibody (HRP‐Ab2) encapsulated on gold nanoparticles caped by cysteamine was immobilized on the surface of GCE modified electrode. A transmission electron microscopy images shows that a sandwich immunoreaction was done and binding of Ab1 and Ab2 performed successfully. Various parameters of immunoassay, including the loading of magnetic nanoparticles, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.001 μg. L^?1 of PSA was obtained under optimum experimental conditions. It is found that such magneto‐bioassay could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for early stage diagnosis of cancer in near future.     

Chemiluminescence selectivity enhancement in the on‐chip Ru(bpy)32+ system: The potential role of buffer type and pH in the determination of hydrochlorothiazide in combined formulations and human plasma

A simple and highly selective on‐chip Ru(bpy)₃²⁺–oxidant chemiluminescence (CL) approach for estimation of a diuretic drug, hydrochlorothiazide (HCZ), in biological fluids was realized in the presence of other fixed‐dose combination drugs by manipulating simultaneously the method of active species (Ru(bpy)₃³⁺) production and type of carrier buffer with pH used for the CL reaction. Chemical oxidation processes involved in the Ru(bpy)₃²⁺–Ce(IV) CL system have been successfully miniaturised in this study using a microfabricated device to generate Ru(bpy)₃³⁺ instantaneously. The proposed system was then screened using HCZ and other drugs in the presence of various buffers and pH to explore the difference in CL emission. Ammonium formate buffer (0.15 M) at pH 4.5 exhibited excellent selectivity towards HCZ when Ru(bpy)₃³⁺ was produced by chemical oxidation using Ce(IV). The newly developed conditions do not involve any kind of prior separation or isolation procedure to remove other combination therapy drugs in formulation and biological samples. The method under fully optimised conditions exhibited wide linearity over the concentration range 0.5–1000 ng ml^?1 and low detection and quantification limits of 0.13 and 0.47 ng ml^?1 respectively for HCZ. Acceptable levels of recoveries were obtained for HCZ from human plasma using the proposed method (98.9–100.8%) in the presence of other antihypertensive combination therapy drugs. This study postulates that such miniaturised devices may find applications especially for on‐site analysis, such as doping control examinations.     

Effect of short‐term (0–72 h) fasting on serum biochemical characteristics in rainbow trout Oncorhynchus mykiss

An experiment was carried out to investigate the effects of short‐term fasting periods on the serum biochemical characteristics of rainbow trout, Oncorhynchus mykiss. For this purpose, fish were fasted 0, 6, 12, 24, 48 and 72 h before blood sampling. Thereafter the serum levels of thyroxine (T₄), 3,5,3′‐triiodothyronine (T₃), cortisol, glucose, lactate, triglyceride, cholesterol, total protein, albumin, globulin and albumin : globulin ration (A : G) were determined. Results show that serum levels of T₄ (4.60–8.77 ng ml^?1), T₃ (7.50–13.3 ng ml^?1), cortisol (7.91–24.5 ng ml^?1), glucose (18.5–80.1 mg dl^?1), lactate (12.7–29.6 mg dl^?1), triglyceride (171–500 mg dl^?1), and cholesterol (321–535 mg dl^?1) were significantly affected by the fasting period. However, there were no significant changes in serum total protein (3.03–3.68 g dl^?1), albumin (1.78–2.01 g dl^?1), globulin (1.15–1.70 g dl^?1) or A : G (1.13–1.93) among the fish fasted 0–72 h. Results clearly show the importance of a fasting period on the serum biochemical properties of rainbow trout. According to the results, 24 h fasting is suggested as a pre‐sampling fasting period in rainbow trout to measure serum levels of T₄, T₃, cortisol, glucose, lactate, triglyceride, and cholesterol. Potential mechanisms related to the changes in biochemical properties are discussed.     

Dithiocarbamate prodrugs activated by prostate specific antigen to target prostate cancer

Disulfiram in conjunction with copper has been shown to be a potent anticancer agent. However, disulfiram’s therapeutic potential in prostate cancer is hindered by off-target effects due to its reactive and nucleophilic thiol-containing component, diethyldithiocarbamate (DTC). To minimize undesirable reactivity, we have strategically blocked the thiol moiety in DTC with a cleavable p-aminobenzyl (pAB) group linked to peptide substrates recognized by prostate specific antigen (PSA). Here we report the synthesis and evaluation in cancer cell models of two PSA-activatable prodrugs: HPD (Ac-HSSKLQL-pAB-DTC and RPD (RSSYYSL-pAB-DTC). In vitro exposure to PSA was found to trigger activation of HPD and RPD to release diethyldithiocarbamate, and both prodrugs were found to induce toxicity in prostate cancer cells, with HPD showing the most promising selectivity. With copper supplementation, the IC₅₀ of HPD was 1.4 µM in PSA-expressing LNCaP cells, and 11 µM in PC3 cells that do not express PSA. These studies demonstrate the utility of using peptide recognition handles to direct the activity of dithiocarbamate prodrugs for selective cytotoxicity of cancer cells.     

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA⁺ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.     

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil

Aims: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. Methods and Results: The antiviral activity was evaluated against human herpesvirus type 1 (HSV‐1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV‐WR) and dengue virus 2 (DENV‐2) by the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti‐HSV‐1 [EC₅₀ 142·8 ± 5·3 μg ml^?1; selectivity index (SI) 2·0] and anti‐DENV‐2 activity (EC₅₀ 9·8 ± 1·3 μg ml^?1; SI 1·5). The pectolinarin ( 1 ) isolated from LEE was less active against HSV‐1 and DENV‐2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV‐1. A mixture of pectolinarin ( 1 ) and acacetin‐7‐O‐rutinoside ( 2 ) was isolated from FEE and has presented anti‐DENV‐2 activity (EC₅₀ 11·1 ± 1·6 μg ml^?1; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. Conclusions: These data shows that D. elongata has antiviral activity mainly against HSV‐1 and DENV‐2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. Significance and Impact of the Study: Distictella elongata might be considered a promising source of anti‐dengue fever phytochemicals.     

High‐performance liquid chromatography study of gatifloxacin and sparfloxacin using erythrosine as post‐column resonance Rayleigh scattering reagent and mechanism study

Herein, a highly selective high‐performance liquid chromatography (HPLC) coupled with resonance Rayleigh scattering (RRS) method was developed to detect gatifloxacin (GFLX) and sparfloxacin (SPLX). GFLX and SPLX were first separated by HPLC, then, in pH 4.4 Britton–Robinson (BR) buffer medium, protonic quaternary ammonia cation of GFLX and SPLX reacted with erythrosine (ERY) to form 1:1 ion‐association complexes, which resulted in a significant enhancement of RRS signal. The experimental conditions of HPLC and post‐column RRS have been investigated, including detection wavelength, flow rate, pH, reacting tube length and reaction temperature. Reaction mechanism were studied in detail by calculating the distribution fraction. The maximum RRS signals for GFLX and SPLX were recorded at λ_ex = λ_em = 330 nm. The detection limits were 3.8 ng ml^?1 for GFLX and 17.5 ng ml^?1 for SPLX at a signal‐to‐noise ratio of 3. The developed method was successfully applied to the determination of GFLX and SPLX in water samples. Recoveries from spiked water samples were 97.56–98.85%.     

Selective recognition of enzymatically active prostate-specific antigen (PSA) by anti-PSA monoclonal antibodies

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.     

Flow‐injection determination of vitamin A in pharmaceutical formulations using Tris(2,2′‐bipyridyl)Ru(II)–Ce(IV) chemiluminescence detection

A flow injection method with chemiluminescence detection is reported for the determination of vitamin A. The method is based on the enhancement effect of vitamin A on chemiluminescence of tris(2,2′‐bipyridyl)Ru(II)–Ce(IV) in acidic medium. The proposed procedure is used to quantitate vitamin A in the range 1.0–100 × 10^?6 mol/L with a correlation coefficient of 0.9991 (n = 9) and relative standard deviation in the range 1.2–2.3% (n = 4). The limit of detection (3 × blank) was 8.0 × 10^?8 mol/L with a sample throughput of 100/h. The effect of common excipients used in pharmaceutical formulations and some clinically important compounds was also studied. The method was applied to determine vitamin A in pharmaceutical formulations and the results obtained were in reasonable agreement with the amount quoted. The results were compared using spectrophotometric method and no significant difference was found between the results of the two methods at 95% confidence limit. Copyright © 2009 John Wiley & Sons, Ltd.