**Validation of a Quantitative Determination Method of Paramino-Salicylic Acid by High-Performance Liquid Chromatography and Its Application in Rat Plasma**

Ibrahima Youm, Malika Lahiani-Skiba and Mohamed Skiba *Laboratoire de Pharmacie Galénique, UMR CNRS 5007 UFR Médecine et Pharmacie, Université de ROUEN, Rouen France* 

#### **1. Introduction**

164 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

[72] McLeod RS, Churchill DN, Lock AM, *et al*. Quality of life of patients with ulcerative colitis preoperatively and postoperatively. *Gastroenterology* 1991; 101: 307-13 [73] Kohler LW, Pemberton JH, Zinsmeister AR, *et al*. Quality of life after proctocolectomy.

[74] Drossman DA, Mitchell CM, Appelbaum MI, *et al*. Do IBD ostomates do better? A

*Gastroenterolgy* 1991; 101: 679-84

1989; 96: 130

A comparison of Brook ileostomy, Kock pouch, and ileal pouch-anal anastomosis.

study of symptoms and health-related quality of life (abstract). *Gastroenterology*

The non steroid anti-inflammatory drugs (NSAID) are among the most prescribed because of their analgesic and anti-inflammatory properties [1].The derivatives of the acid aminosalicylic (4ASA and 5-ASA) are used since many years in the treatment of the intestinal chronic inflammatory diseases: Crohn disease and colitis. Aminosalicylic acids exert a direct anti- inflammatory action on the intestinal mucous membranes. They do not have any link with the drugs of cortisone family, or with traditional NSAID used for pain and rheumatism. They are also different from the acetylsalicylic acid (Aspirine®).

4ASA also known under the name of paramino benzoïc acid (PABA) or paramino salicylic acid (PASA) (figure 1) is employed for the treatment of the ulcerative colitis and Crohn disease. These diseases are characterized by an ignition of the colorectal mucous membrane [2].

Fig. 1. Molecular structure of 4ASA

Validation of a Quantitative Determination Method of Paramino-Salicylic

**2.3.2 Linearity** 

day variation.

**2.3.3 Accuracy** 

**2.3.4 Robustness** 

factorial design was used (Table **1**).

pH on the robustness of the method:

and the slope of the calibration curve.

**2.3.5 Limit of detection and limit of quantification** 

and pH.

Acid by High-Performance Liquid Chromatography and Its Application in Rat Plasma 167

Data were obtained using a stock solution of 4ASAS at 0.5 mg.ml-1 as concentration. This solution was then diluted using highly purified water to obtain the standard solutions from 1 to 125µg/ml concentrations. Five injections were made for each concentration. The linearity of the calibration curves was determined on three different days for intra or inter-

Accuracy of the proposed method was established by recovery experiments using standard addition method. This study was employed by addition of a standard reference (pure marketed product whose purity was tested beforehand by Differential Scanning

An experimental design was used to evaluate the influence of selected factors. Two-level

Table 1. Influence of selected factors: MeCN (%), Wavelength (nm), Flow rate (ml/min) and

For this method only factors were examinated: acetonitrile ratio, wavelength, flow rate

Several approaches were used to determine the detection and quantitation limits. These include visual evaluation, signal-to-noise ratio, the use of standard deviation of the response

In the present study, the LOD was related to signal / noise of the system and was defined as a peak whose report/ration signal background noise was at least equal to 3/1. The LOQ was defined as a peak whose report/ ratio signal background noise was at least equal to 10/1.

Calorimetry (DSC) and Fourier transformed Infrared Spectrometry (FT-IR).

In the literature, several analytical methods primarily based on separation were described. Therefore, quantitative methods to assay, for instance a drug substance or its impurities, have needed to be fully validated after development. Depending on the type of analytical method and its intended use, a check for linearity, precision, accuracy, specificity, range, limits of detection and quantification, and/or robustness, is required [3-7], gas chromatography coupled with the mass spectrometry were the most usually used methods for analytes separation [8]. A capillary method of electrophoresis was developed for 4ASA determination and its metabolites: acid N-acetyl-p-aminosalicylic (N-acetyl-PASA) in urine. Good separation of analytes was carried out with a 12 min retention time for15min [9]. 4ASA is also quantified by HPLC in the biological fluids [10, 11]. In this present article, we describe the development of a new RP-HPLC method for the determination of 4ASA at the same time in both aqueous medium and plasma.

## **2. Material and methods**

4ASA was supplied by Merck (Hohenbrunn, Germany). Formic acid (99-100%) was provided by Prolabo. Potassium dihydrogene phosphate (KH2PO4), acetonitrile (MeCN) and potassium hydroxide (KOH) were provided by SDS (Peypin, France). Alpha cyclodextrine was purchased from Waker and Gelucire® from Gattefossé (France). Sodium citrate was obtained from Sigma chemical Co (USA). Water purified on Milli-Q system (Millipore, USA) was used. All other chemicals were of analytical grade.

#### **2.1 Instrumentation**

The liquid chromatographic system used in the present study, consisted of a pump (880- Jasco, Japan), an automatic injector (autosampler HPLC -360Kontron, Brehme, Germany) and a detector (Jasco 875-UV). All the parameters of HPLC were controlled by the Azur software: version 3.0 coupled to an acquisition box (Azur PAD).

## **2.2 Chromatographic conditions**

The mobile phase was prepared by mixing water, formic acid and acetonitrile in varied proportions. The optimum mobile phase used in the validation studies consisted of waterformic acid-acetonitrile 67:3:30, (v/v/v). Before analysis the mobile phase was filtered through a 0.45µm membrane and degassed by ultra sonication (Transsonic 950, Prolabo). Solvent delivery was employed at a flow rate of 1.0 ml min -1. A Kromasil column: C-18µm, 250mm ×4.6mm (Chromato, France HAS) was used and maintained at room temperature. Detection of the analytes was carried out at 300nm.The appropriate wavelength for the detection of the drug was determined by wavelength scanning over the range of 200-400nm. Injection volume of the analytes was set to a constant volume of 50µl.

#### **2.3 Validation of the method**

The described method has been validated in terms of specificity, precision, linearity, accuracy, limit of detection (LOD) and limit of quantification (LOQ).

#### **2.3.1 Precision**

Precision was expressed with respect to the intra and inter-day variations in the expected drug concentrations.

#### **2.3.2 Linearity**

166 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

In the literature, several analytical methods primarily based on separation were described. Therefore, quantitative methods to assay, for instance a drug substance or its impurities, have needed to be fully validated after development. Depending on the type of analytical method and its intended use, a check for linearity, precision, accuracy, specificity, range, limits of detection and quantification, and/or robustness, is required [3-7], gas chromatography coupled with the mass spectrometry were the most usually used methods for analytes separation [8]. A capillary method of electrophoresis was developed for 4ASA determination and its metabolites: acid N-acetyl-p-aminosalicylic (N-acetyl-PASA) in urine. Good separation of analytes was carried out with a 12 min retention time for15min [9]. 4ASA is also quantified by HPLC in the biological fluids [10, 11]. In this present article, we describe the development of a new RP-HPLC method for the determination of 4ASA at

4ASA was supplied by Merck (Hohenbrunn, Germany). Formic acid (99-100%) was provided by Prolabo. Potassium dihydrogene phosphate (KH2PO4), acetonitrile (MeCN) and potassium hydroxide (KOH) were provided by SDS (Peypin, France). Alpha cyclodextrine was purchased from Waker and Gelucire® from Gattefossé (France). Sodium citrate was obtained from Sigma chemical Co (USA). Water purified on Milli-Q system

The liquid chromatographic system used in the present study, consisted of a pump (880- Jasco, Japan), an automatic injector (autosampler HPLC -360Kontron, Brehme, Germany) and a detector (Jasco 875-UV). All the parameters of HPLC were controlled by the Azur

The mobile phase was prepared by mixing water, formic acid and acetonitrile in varied proportions. The optimum mobile phase used in the validation studies consisted of waterformic acid-acetonitrile 67:3:30, (v/v/v). Before analysis the mobile phase was filtered through a 0.45µm membrane and degassed by ultra sonication (Transsonic 950, Prolabo). Solvent delivery was employed at a flow rate of 1.0 ml min -1. A Kromasil column: C-18µm, 250mm ×4.6mm (Chromato, France HAS) was used and maintained at room temperature. Detection of the analytes was carried out at 300nm.The appropriate wavelength for the detection of the drug was determined by wavelength scanning over the range of 200-400nm.

The described method has been validated in terms of specificity, precision, linearity,

Precision was expressed with respect to the intra and inter-day variations in the expected

(Millipore, USA) was used. All other chemicals were of analytical grade.

software: version 3.0 coupled to an acquisition box (Azur PAD).

Injection volume of the analytes was set to a constant volume of 50µl.

accuracy, limit of detection (LOD) and limit of quantification (LOQ).

the same time in both aqueous medium and plasma.

**2. Material and methods** 

**2.1 Instrumentation** 

**2.2 Chromatographic conditions** 

**2.3 Validation of the method** 

**2.3.1 Precision** 

drug concentrations.

Data were obtained using a stock solution of 4ASAS at 0.5 mg.ml-1 as concentration. This solution was then diluted using highly purified water to obtain the standard solutions from 1 to 125µg/ml concentrations. Five injections were made for each concentration. The linearity of the calibration curves was determined on three different days for intra or interday variation.

## **2.3.3 Accuracy**

Accuracy of the proposed method was established by recovery experiments using standard addition method. This study was employed by addition of a standard reference (pure marketed product whose purity was tested beforehand by Differential Scanning Calorimetry (DSC) and Fourier transformed Infrared Spectrometry (FT-IR).

## **2.3.4 Robustness**

An experimental design was used to evaluate the influence of selected factors. Two-level factorial design was used (Table **1**).



Table 1. Influence of selected factors: MeCN (%), Wavelength (nm), Flow rate (ml/min) and pH on the robustness of the method:

For this method only factors were examinated: acetonitrile ratio, wavelength, flow rate and pH.

## **2.3.5 Limit of detection and limit of quantification**

Several approaches were used to determine the detection and quantitation limits. These include visual evaluation, signal-to-noise ratio, the use of standard deviation of the response and the slope of the calibration curve.

In the present study, the LOD was related to signal / noise of the system and was defined as a peak whose report/ration signal background noise was at least equal to 3/1. The LOQ was defined as a peak whose report/ ratio signal background noise was at least equal to 10/1.

The determination of these values was made starting from the line of the range standard. These two values were calculated from the Response Factor (Rf) according to formulas (1) and (2): the initial concentration corresponds to the intersection of the right-hand side to the y-axis [12].

$$\text{Fr} = \frac{\text{Standard area}}{\text{Initial Concentration}} \tag{1}$$

Validation of a Quantitative Determination Method of Paramino-Salicylic

room temperature for ten minutes and analyzed by HPLC.

**3.4 Blood-plasma partition ratio** 

**4. Results and discussion** 

**4.2. Validation of the method 4.2.1 Specificity/selectivity** 

linear within the concentration range (Figure **2**).

**4.1 Chromatographic conditions** 

4ASA oral doses to rats.

**3.5 Bioavailability** 

30 min.

300nm.

formulation.

**4.2.2 Linearity** 

quantified using

Acid by High-Performance Liquid Chromatography and Its Application in Rat Plasma 169

Blood-plasma partition ratio would predict plasma concentration-time courses following

Partition test was carried out using three fresh samples of blood (1ml) mixed with 500µl of 100µg/ml standard solution of 4ASA. The mixture was maintained in constant stirring at

Each rat received an oral dose of minigranules containing 25mg of 4ASA. Three hours after administration of minigranules, the rats were anesthetized with an intra peritoneal pentobarbital 10% (ABBOT, Ringis, France) at the dose of 100µl/100g (re-injected with 10µl/100g as necessary). The internal carotid artery was cannulated via 4cm paratracheal

Rats were killed at the end of experiments by exsanguinations. These samples were collected into eppendorf tubes containing 500µl of citrate buffer (0.129M) and kept on ice for at least

Plasma was obtained by centrifugation (Eppendorf centrifuge, Germany) at 3500 rpm for 10 min and transferred to clean test tubes. After vortexing for 1 min using a Heidolph Top-mix 94323 (Bioblock scientific, Germany), plasma samples were analyzed by HPLC method.

Experiments were carried out with 10 up to 90% of acetonitrile and water as a mobile phase. The best peak shape and the maximum of separation were obtained with a binary mixture: water - acetonitrile (70% and 30% respectively). A minimum of retention time (Rt=4.3-4.8)

The optimum wavelength for analyte detection with adequate sensitivity was found to be

A chromatogram was obtained from blank samples. No peak corresponding to the endogenous compounds was detected at the analyte retention time (4.3-4.8min) at 300nm. It means that the developed method is selective in relation of the used carrier for pellet

Seven-point linearity curve was constructed for three consecutive days. Samples were

concentrations – peak area relationships and were calculated by the simple regression analysis. The plots of peak area ratios versus concentrations of all analytes were found to be

incision, and blood samples were taken at 0.5, 1, 2, 3 and 4 hours after anesthesia.

Analyte concentrations were calculated by using an account of the factor of dilution.

with good peak resolution was obtained under a flow of 1ml.min1.

Retention time was very sensitive to the freshness or ageing of mobile phase.

$$\text{Sample concentration} = \frac{\text{Sample area}}{Fr} \tag{2}$$

## **2.3.6 Stability**

Experiments were carried out under the same conditions as the samples analysis.

## **2.3.7 Recovery test**

Recovery test was carried out by weighing three batches of pellets (containing 50% of 4ASA) compared with a powder sample of pure active ingredient. Amounts representing 120,100 and 80% of 4ASA were used as equivalent concentrations. Each batch is crushed using a mortar then filtered before being solubilized in 250ml water with respect of sink conditions. The release properties of the active ingredient from the extruded formulations matrix were studied by using a process of the delayed release. The medium of dissolution was maintained at 37°C with 100 rpm stirring. After 6 hours, three samples of 1 ml were taken in each sample of buffer solution of phosphate potassium (0.2 M) whose pH was adjusted to 7.5 using a potassium hydroxide buffer (KOH 1M).

## **3. Application to biological samples**

The proposed method was applied to the determination of 4ASA in plasma samples from the bioequivalence study. *In vivo* studies of 4ASA blood concentration were carried out by using established methods of the literature [10]. Male Sprague-Dawley rats weighing 200– 250g (*n* = 4) were purchased from Charles Rivers Enterprise (France) and kept in laboratory conditions before experiments.

## **3.1 Linearity and precision**

Linearity was assessed by analyzing seven standards with concentrations over the range of 10-100µg/ml in plasma (n=5). Precision and accuracy were tested comparing to the values obtained previously in aqueous solution. Aliquot of 1000µl plasma were mixed with 500µl of standard solution of 4ASA. The mixture was vortexed for 2 min and transferred into the vial for HPLC analysis (n=8).

#### **3.2 Recovery test in plasma**

For recovery test, three samples of 1000µl plasma were mixed with 250µl of standard solution (25, 50 and 100µg/ml) of 4ASA. The mixture was vortexed for 2 min and transferred into the vial for HPLC analysis (n=5).

#### **3.3 Stability test in plasma**

Drug stability during sample collection and processing is an important factor for clinical bioanalysis. Three samples of 1000µl plasma were mixed with 500µl of standard solution of 4ASA and analyzed at 0 and 24hours at room temperature and at 4°C (n=5).

## **3.4 Blood-plasma partition ratio**

Blood-plasma partition ratio would predict plasma concentration-time courses following 4ASA oral doses to rats.

Partition test was carried out using three fresh samples of blood (1ml) mixed with 500µl of 100µg/ml standard solution of 4ASA. The mixture was maintained in constant stirring at room temperature for ten minutes and analyzed by HPLC.

## **3.5 Bioavailability**

168 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

The determination of these values was made starting from the line of the range standard. These two values were calculated from the Response Factor (Rf) according to formulas (1) and (2): the initial concentration corresponds to the intersection of the right-hand side to the

������� ������������� (1)

�� (2)

Fr = �������� ����

7.5 using a potassium hydroxide buffer (KOH 1M).

**3. Application to biological samples**

conditions before experiments.

**3.1 Linearity and precision** 

for HPLC analysis (n=8).

**3.2 Recovery test in plasma** 

**3.3 Stability test in plasma** 

transferred into the vial for HPLC analysis (n=5).

Sample concentration = ������ ����

Experiments were carried out under the same conditions as the samples analysis.

Recovery test was carried out by weighing three batches of pellets (containing 50% of 4ASA) compared with a powder sample of pure active ingredient. Amounts representing 120,100 and 80% of 4ASA were used as equivalent concentrations. Each batch is crushed using a mortar then filtered before being solubilized in 250ml water with respect of sink conditions. The release properties of the active ingredient from the extruded formulations matrix were studied by using a process of the delayed release. The medium of dissolution was maintained at 37°C with 100 rpm stirring. After 6 hours, three samples of 1 ml were taken in each sample of buffer solution of phosphate potassium (0.2 M) whose pH was adjusted to

The proposed method was applied to the determination of 4ASA in plasma samples from the bioequivalence study. *In vivo* studies of 4ASA blood concentration were carried out by using established methods of the literature [10]. Male Sprague-Dawley rats weighing 200– 250g (*n* = 4) were purchased from Charles Rivers Enterprise (France) and kept in laboratory

Linearity was assessed by analyzing seven standards with concentrations over the range of 10-100µg/ml in plasma (n=5). Precision and accuracy were tested comparing to the values obtained previously in aqueous solution. Aliquot of 1000µl plasma were mixed with 500µl of standard solution of 4ASA. The mixture was vortexed for 2 min and transferred into the vial

For recovery test, three samples of 1000µl plasma were mixed with 250µl of standard solution (25, 50 and 100µg/ml) of 4ASA. The mixture was vortexed for 2 min and

Drug stability during sample collection and processing is an important factor for clinical bioanalysis. Three samples of 1000µl plasma were mixed with 500µl of standard solution of

4ASA and analyzed at 0 and 24hours at room temperature and at 4°C (n=5).

y-axis [12].

**2.3.6 Stability** 

**2.3.7 Recovery test** 

Each rat received an oral dose of minigranules containing 25mg of 4ASA. Three hours after administration of minigranules, the rats were anesthetized with an intra peritoneal pentobarbital 10% (ABBOT, Ringis, France) at the dose of 100µl/100g (re-injected with 10µl/100g as necessary). The internal carotid artery was cannulated via 4cm paratracheal incision, and blood samples were taken at 0.5, 1, 2, 3 and 4 hours after anesthesia.

Rats were killed at the end of experiments by exsanguinations. These samples were collected into eppendorf tubes containing 500µl of citrate buffer (0.129M) and kept on ice for at least 30 min.

Plasma was obtained by centrifugation (Eppendorf centrifuge, Germany) at 3500 rpm for 10 min and transferred to clean test tubes. After vortexing for 1 min using a Heidolph Top-mix 94323 (Bioblock scientific, Germany), plasma samples were analyzed by HPLC method. Analyte concentrations were calculated by using an account of the factor of dilution.

## **4. Results and discussion**

#### **4.1 Chromatographic conditions**

Experiments were carried out with 10 up to 90% of acetonitrile and water as a mobile phase. The best peak shape and the maximum of separation were obtained with a binary mixture: water - acetonitrile (70% and 30% respectively). A minimum of retention time (Rt=4.3-4.8) with good peak resolution was obtained under a flow of 1ml.min1.

The optimum wavelength for analyte detection with adequate sensitivity was found to be 300nm.

Retention time was very sensitive to the freshness or ageing of mobile phase.

## **4.2. Validation of the method**

## **4.2.1 Specificity/selectivity**

A chromatogram was obtained from blank samples. No peak corresponding to the endogenous compounds was detected at the analyte retention time (4.3-4.8min) at 300nm. It means that the developed method is selective in relation of the used carrier for pellet formulation.

## **4.2.2 Linearity**

Seven-point linearity curve was constructed for three consecutive days. Samples were quantified using

concentrations – peak area relationships and were calculated by the simple regression analysis. The plots of peak area ratios versus concentrations of all analytes were found to be linear within the concentration range (Figure **2**).

Validation of a Quantitative Determination Method of Paramino-Salicylic

quantification (305ng/ml).

analyte amount (Figure **3**).

**4.2.5 Recovery test**

**4.2.6 Robustness** 

**AUC** 

have any effect on the robustness (Figure 4).

modify the analyte protonation.

**4.2.7 Study of 4ASA stability** 

**4.2.4 Limit of Detection (LOD) and Limit of Quantification (LOQ)** 

Acid by High-Performance Liquid Chromatography and Its Application in Rat Plasma 171

The results showed a less significant limit of detection (105ng/ml) compared to the limit of

The overall mean recoveries calculated were 80, 100 and 120 for low, medium and high quality control samples respectively. A linear curve was obtained meaning proportional

**Recovery test**

y = 933.02x + 217.12 R² = 0.9577

Fig. 3. Chart of the test of recovering: Bars represent standard deviations of the mean (n=9).

0% 50% 100% 150%

**Concentration**

Two–level fractional factorial design were applied in this method. The operational parameters do not lead to essential changes of the performance of the chromatographic system except the wavelength. Only the wavelength seemed to influence the robustness of the method. The interaction between the acetonitrile ratio and the flow rate didn't seem to

The obtained values were accurate and RSD values didn't exceed 3.3%. On the other hand the retention time varied quickly from 2.98 to 5.28 min for each modified parameter. Acetonitrile change ratio would change the polarity of the mobile phase while pH could

Three aliquots were used during this experiment which lasted 6 hours. The medium of dissolution was maintained with 37°C, in constant stirring with 100 rpm to simulate gastrointestinal conditions. The results showed a relative stability of 93.5% (Figure **5**).

Fig. 2. Linearity of quantitative method by HPLC. Bars represent standard deviations of the mean (n = 5)

#### **4.2.3 Accuracy and precisions**

The results were expressed as percent recoveries of the particular components in the samples. The average percent recoveries (±RSD) were respectively in the range of 97.3±0.9 - 99±1.7 (Table **2**).


Table 2. Accuracy of the proposed method (n =10). AUC: Area Under Curve

The results of intra-day and inter-day precision experiments are indicated according to the averages and relative standard deviations (RSD). Inter-day and intra-day precision given by relative standard deviation (%R.S.D.) of quality control samples were ≤ 3.3%. The calculated values are presented onto (Table **3**).


Table 3. Intra and inter-day precision of 4ASA quantification with area (UA) andRelative Standard Deviation (RSD) (n=3).

## **4.2.4 Limit of Detection (LOD) and Limit of Quantification (LOQ)**

The results showed a less significant limit of detection (105ng/ml) compared to the limit of quantification (305ng/ml).

## **4.2.5 Recovery test**

170 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Calibration curve

y = 102.4x + 26.789 R² = 0.9984

Fig. 2. Linearity of quantitative method by HPLC. Bars represent standard deviations of the

0 10 20 30 40 50 60

Concentration (µg/ml)

The results were expressed as percent recoveries of the particular components in the samples. The average percent recoveries (±RSD) were respectively in the range of 97.3±0.9 -

> **Concentration (mg/ml) 0,005 0,0075 0,01 Mean of AUC 329,747 489,049 725,458 RSD (%) 0,740 1,874 0,51**

The results of intra-day and inter-day precision experiments are indicated according to the averages and relative standard deviations (RSD). Inter-day and intra-day precision given by relative standard deviation (%R.S.D.) of quality control samples were ≤ 3.3%. The calculated

Table 3. Intra and inter-day precision of 4ASA quantification with area (UA) andRelative

Table 2. Accuracy of the proposed method (n =10). AUC: Area Under Curve

mean (n = 5)

99±1.7 (Table **2**).

**4.2.3 Accuracy and precisions** 

AUC

values are presented onto (Table **3**).

Standard Deviation (RSD) (n=3).

The overall mean recoveries calculated were 80, 100 and 120 for low, medium and high quality control samples respectively. A linear curve was obtained meaning proportional analyte amount (Figure **3**).

Fig. 3. Chart of the test of recovering: Bars represent standard deviations of the mean (n=9).

## **4.2.6 Robustness**

Two–level fractional factorial design were applied in this method. The operational parameters do not lead to essential changes of the performance of the chromatographic system except the wavelength. Only the wavelength seemed to influence the robustness of the method. The interaction between the acetonitrile ratio and the flow rate didn't seem to have any effect on the robustness (Figure 4).

The obtained values were accurate and RSD values didn't exceed 3.3%. On the other hand the retention time varied quickly from 2.98 to 5.28 min for each modified parameter. Acetonitrile change ratio would change the polarity of the mobile phase while pH could modify the analyte protonation.

#### **4.2.7 Study of 4ASA stability**

Three aliquots were used during this experiment which lasted 6 hours. The medium of dissolution was maintained with 37°C, in constant stirring with 100 rpm to simulate gastrointestinal conditions. The results showed a relative stability of 93.5% (Figure **5**).

Validation of a Quantitative Determination Method of Paramino-Salicylic

The validated analytical method was applied to male Sprague-Dawley rats.

the specificity of the drug.

calibrations standards.

separate degradation products for pure drug.

Table 4. Evaluation of the Intra assay precision (n=8)

4ASA reached a maximum of 101.95% (Figure 6).

Fig. 6. Plasmatic concentration of 4ASA after partition-ratio.

Bars represent standard deviations (n=5)

**Concentration (µg/ml) ±RSD**

**4.3 Application to biological samples** 

Acid by High-Performance Liquid Chromatography and Its Application in Rat Plasma 173

The determination of 4ASAconcentration following the degradation, was a positive point on

It would mean that the first breackdowned products did not absorb at 300nm. However new peaks at lower retention time (<3min) were detected on seven day-old samples at 300nm.The later consideration deals with the specificity of the method and its ability to

Compared to the results given by working standard solutions, good peak shape and acceptable sensitivity were obtained with the same mobile phase (acetonitrile /water 30:70) from diluted plasma samples containing 4ASA. Good precision (CV=3.39%) and reproducibility were also observed during 4ASA quantification in plasma samples (Table **4**)**.**

> Area % Mean 8849,978 100,7479 RSD 152,8166 2,718627

The accuracy of these values was confirmed by back calculating the concentration of the

The retention time was from 4.3 to 5.2 min and did not interfer with other compounds from plasma. This period was closed to that obtained with those obtained with aqueous solution (4.3-4.8min). The concentration range (10-100µg/ml) in plasma is shorter than the aqueous medium concentration (1 to 125µg/ml). The regression equation was Y=88.72 x-88.38 and coefficient of correlation (*r2*) was 0,999.With regard to the partition test, plasma level of

**Partition-ratio : plasmatic concentration of 4-ASA**

Control 1 2 3 **Sample**

Fig. 4. Histogram representing robustness test of the method. Bars represent standard deviations of the mean (n=5).

Fig. 5. Study of 4ASA stability during 6h. Bars represent standard deviations of the mean (n=3)

The determination of 4ASAconcentration following the degradation, was a positive point on the specificity of the drug.

It would mean that the first breackdowned products did not absorb at 300nm. However new peaks at lower retention time (<3min) were detected on seven day-old samples at 300nm.The later consideration deals with the specificity of the method and its ability to separate degradation products for pure drug.

### **4.3 Application to biological samples**

172 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

MeCN (%)

WL (Nm)

pH

Débit(ml/min)

Robustness

Fig. 4. Histogram representing robustness test of the method. Bars represent standard


**Effect ±RSD** 

Fig. 5. Study of 4ASA stability during 6h. Bars represent standard deviations of the mean (n=3)

0123456

Time (h)

**Stability test of 4-ASA**

deviations of the mean (n=5).

80

90

100

Drug in percent

110

**Parameters**

The validated analytical method was applied to male Sprague-Dawley rats.

Compared to the results given by working standard solutions, good peak shape and acceptable sensitivity were obtained with the same mobile phase (acetonitrile /water 30:70) from diluted plasma samples containing 4ASA. Good precision (CV=3.39%) and reproducibility were also observed during 4ASA quantification in plasma samples (Table **4**)**.**



The accuracy of these values was confirmed by back calculating the concentration of the calibrations standards.

The retention time was from 4.3 to 5.2 min and did not interfer with other compounds from plasma. This period was closed to that obtained with those obtained with aqueous solution (4.3-4.8min). The concentration range (10-100µg/ml) in plasma is shorter than the aqueous medium concentration (1 to 125µg/ml). The regression equation was Y=88.72 x-88.38 and coefficient of correlation (*r2*) was 0,999.With regard to the partition test, plasma level of 4ASA reached a maximum of 101.95% (Figure 6).

Fig. 6. Plasmatic concentration of 4ASA after partition-ratio. Bars represent standard deviations (n=5)

Validation of a Quantitative Determination Method of Paramino-Salicylic

7.5h. Bars represent standard deviations of the mean (n=4).

that this method is suitable for pharmacokinetic studies.

The authors thank also In-Cyclo Society for his financial support.

[2] GETAID. Intest info "les derives amino-salicylés"., 2005

[1] Wolfe, M.M.; Lichtenstein, D.R, Singh, G. *N Engl J Med.,* 1999, 340, 1888–99.

[5] Sadeg, N.; Pertat, N.; Dutertre, H.; Dumontet, M. J. Chromatogr., 1996, 675,113.

[3] Defilippi, A.; Piancone, G.; Costa Laia, R. Tibaldi, G.P. Chromatographia., 1995, 40-170. [4] Defilippi, A.; Piancone, G.; Costa Laia, R. ; Balla, S.; Tibaldi, G.P. J. Chromatogr., 1994,

**5. Conclusion** 

0 0.5 1 1.5 2 2.5 3 3.5 4

Concentration (µg/ml) ±RSD

**6. Acknowledgements** 

656-466.

**7. References** 

Acid by High-Performance Liquid Chromatography and Its Application in Rat Plasma 175

Bioavailability of 4-ASA

Fig. 8. Plasma concentration-time profile after i.v. administration of 25 mg/kg 4- ASA after

3 3.5 4 5 6 7 7.5

Hours (h)

In pharmaceutical analysis, often a method validation is required in order to meet the strict regulations, set by the regulatory authorities**.** This method was then developed and validated for the determination of 4ASA. Thus specificity, precision, accuracy, linearity, recovery and the limits were acceptable with the execution run time for control studies. It would be very useful for release profile study in simulated intestinal medium of the analyte in question. These results indicated that the RP-HPLC method was specific for the

Through this method the retention time was reduced considerably compared to previous results obtained by Gennaro *et al* [13]. With these present results it is possible to save time and solvents used to constitute the mobile phase. In addition, studies in plasma indicated

determination of 4ASA in plasma, under the chromatographic conditions employed.


The average recovery of assay was 93.54%, 100.89% and 100.23% for low, medium and high concentrations of 4ASA in plasma respectively (Table **5**).

Table 5. Responses in recovery tests at low, mean and high value. Mean ±RSD

Stability tests were investigated using the procedure described above. The results indicated that 4ASA was stable in plasma at -4°C for 24h (Figure **7**).

Fig. 7. Stability test of 4ASA in plasma after 48h at room temperature (RT) and at 4°C. Bars represent standard deviations of the mean (n=5).

Since 4ASA was degraded in human plasma in 24 hours at room temperature and would be useful for immediate-release solid oral dosage forms. The profile of bioavailability test curve was characteristic of a dosage form with extended release. Because the plasma content of 4ASA was almost constant, it could mean that equilibrium of the balance absorption/ degradation was maintained (Figure **8**).

We notice that stability of the analyte during sample collection was investigated using the procedure described above. In connection with bioavailability, these results were unexpected because the concentration remains relatively constant.

Fig. 8. Plasma concentration-time profile after i.v. administration of 25 mg/kg 4- ASA after 7.5h. Bars represent standard deviations of the mean (n=4).

## **5. Conclusion**

174 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

The average recovery of assay was 93.54%, 100.89% and 100.23% for low, medium and high

Stability tests were investigated using the procedure described above. The results indicated

**Stability test of 4-ASA in plasma**

Fig. 7. Stability test of 4ASA in plasma after 48h at room temperature (RT) and at 4°C.

Since 4ASA was degraded in human plasma in 24 hours at room temperature and would be useful for immediate-release solid oral dosage forms. The profile of bioavailability test curve was characteristic of a dosage form with extended release. Because the plasma content of 4ASA was almost constant, it could mean that equilibrium of the balance absorption/

**24 h 48h** 

TA 4°C

control

We notice that stability of the analyte during sample collection was investigated using the procedure described above. In connection with bioavailability, these results were

Bars represent standard deviations of the mean (n=5).

unexpected because the concentration remains relatively constant.

degradation was maintained (Figure **8**).

0

20

40

60

**Concentration (µg/ml)±RSD**

80

100

120

Expected Measured 25 23,39 1,74 50 51,45 3,28 100 100,3 1,65

Concentration (µg/ml) RSD (%)

Table 5. Responses in recovery tests at low, mean and high value. Mean ±RSD

concentrations of 4ASA in plasma respectively (Table **5**).

that 4ASA was stable in plasma at -4°C for 24h (Figure **7**).

In pharmaceutical analysis, often a method validation is required in order to meet the strict regulations, set by the regulatory authorities**.** This method was then developed and validated for the determination of 4ASA. Thus specificity, precision, accuracy, linearity, recovery and the limits were acceptable with the execution run time for control studies. It would be very useful for release profile study in simulated intestinal medium of the analyte in question. These results indicated that the RP-HPLC method was specific for the determination of 4ASA in plasma, under the chromatographic conditions employed.

Through this method the retention time was reduced considerably compared to previous results obtained by Gennaro *et al* [13]. With these present results it is possible to save time and solvents used to constitute the mobile phase. In addition, studies in plasma indicated that this method is suitable for pharmacokinetic studies.

## **6. Acknowledgements**

The authors thank also In-Cyclo Society for his financial support.

## **7. References**


**10** 

Flavio M. Habal

*Canada*

*University Health Network University Of Toronto* 

**Approach to the Management of the** 

**Patient: Successful Outcome** 

**Pregnant Inflammatory Bowel Disease** 

Inflammatory bowel disease (IBD) encompasses Crohn's disease (CD) and ulcerative colitis (UC) and it affect young adults in their reproductive years (Andres & Friedman,

Approximately 25% of these patients will conceive after the diagnosis is made. Many women with IBD raise concerns related to the effects of both the disease and medical therapy on fertility, pregnancy and foetal outcomes. On the basis of prospective and registry data, it is known that women with IBD have a higher risk of low-birth-weight deliveries and pre-term deliveries (Cornish J, 2007). With the exception of patients who have had previous pelvic surgery, the majority of these patients are able to conceive, have a normal pregnancy and a good foetal outcome. Maintaining remission, with drug therapy, prior to conception and during pregnancy is of prime importance. Control of disease activity before conception and during pregnancy is critical to optimize both maternal and fetal health (Mountfield, 2010). Normal maternal weight gain during pregnancy appears to protect against adverse outcome. The majority of drugs used in IBD appear to be safe and their benefit appears to outweigh the risk of disease exacerbation and poor foetal outcome. The newer biologic anti

Although there are reports of some traces of drugs in breast milk in women taking

Treatment of IBD patients who are contemplating conception should be tailored to each patient, taking into accounts the patient wishes and concerns. The patient should be followed by a multidisciplinary team including the family physician, the gastroenterologist

Preconception care aims to ensure the optimal physical and mental well-being of women and their partners at the onset of and during early pregnancy, to increase the likelihood of normal pregnancy and the delivery of a healthy infant. Pre pregnancy counselling should be an integral part of IBD consultation. Patient understanding of the disease and its changes during pregnancy and breastfeeding is vital for a successful outcome. In a recent

TNF alpha drugs appear to be safe during pregnancy.

medications no major undue complication has been reported.

**1. Introduction** 

and the obstetrician.

**Preconception advice and counselling** 

1999).

