VERALIPRIDE/VERALIPRIDA "PARA LOS ENTENDIDOS"

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UNIVERSIDAD DEL EL CAIRO

cromatografía de capa fina - cromatografía de columna de líquido - Los estudios de estabilidad - veraliprida

Dos métodos cromatográficos sensibles y selectivos se han desarrollado y validado para la determinación de veraliprida en presencia de sus productos de degradación. Los estudios se llevaron a cabo la degradación forzada, con HCl, NaOH y 3% H2O2. El primer método se basa en la separación cromatográfica en capa fina del punto de drogas intacta de su degradación, seguido por las mediciones de densitometría. El segundo método se basa en la separación isocrática de cromatografía líquida de la droga estudiada de su degradación en una columna de fase inversa C18. La propuesta de método de LC se utilizó para investigar la cinética del proceso de degradación alcalina del fármaco seleccionado a diferentes temperaturas.
Veraliprida (Ver) es un medicamento anti-psicótico, que se utiliza en el tratamiento de los síntomas cardiovasculares y psicológicos asociados con la menopausia [1-3]. Es la N-[(1-alil-2-pirrolidinilo) metil]-5-sulphamoyl-2-veretramide [4].

En la literatura, la cromatografía de gases con espectrometría de masas o un detector de ionización de llama fue utilizado [5]. La Conferencia Internacional de Armonización (ICH), las directrices recomiendan que las pruebas de estrés deben llevarse a cabo para dilucidar las características inherentes a la estabilidad de la sustancia activa. Ácidos, alcalinos y estabilidad a la oxidación se requiere [6, 7]. En ausencia de métodos han sido publicados para la determinación de Ver en presencia de sus productos de degradación, el objetivo de este trabajo fue desarrollar densitométrico de TLC y los métodos de LC para la determinación de Ver en presencia de sus productos de degradación (Cuadro 1). Además, el método desarrollado LC se utilizó para investigar la cinética del proceso de degradación alcalina.
Tabla 1 Resultados de la validación de los análisis obtenidos mediante la aplicación de la propuesta de TLC y los métodos de LC para la determinación de veraliprida en el fármaco y medicamento.

Parameters Veralipride
TLC LC
Linearity range 1–10 μg spot−1 1–100 μg mL−1
Accuracya
Mean ± RSD%b 99.83 ± 1.44 100.35 ± 1.45
Mean ± RSD%c 99.18 ± 0.676 99.58 ± 1.17
Precision
Intraday RSD%d 0.76 1.52
Interday RSD%d 0.49 0.54
LOD 0.041 μg spot−1 0.015 μg mL−1
LOQ 0.086 μg spot−1 0.044 μg mL−1
Specificity
Mean ± RSD% 100.39 ± 1.10 99.31 ± 1.08
Regression equatione
Slope 1.73 × 102 12.5 × 103
SE of the slope 2.55 83.21
Confidence limit of slopef 1.66–1.80 × 102 12.39–12.79 × 103
Intercept 9.29 × 102 11.80 × 103
SE of the intercept 15.49 4.57 × 103
Confidence limit of interceptf 8.86–9.72 × 102 10.55–22.67 × 103
Correlation coefficient (r) 0.999 0.999
a n = 6
bDrug sustancia
cDrug producto
d n = 9
e Y = a + bc, donde C es la concentración en microgramos terreno para un TLC y en mg mL-1 para LC y Y es el área del pico
f95% intervalo de confianza.
Dos experimentales

2.1 Instrumentación

placas de TLC (20 × 20 cm2, placas de aluminio recubiertas con 0,2 mm F254 de gel de sílice), fueron adquiridos de Macherey Nagel (Alemania) y las muestras se aplica a las placas con un 25 l Hamilton microjeringa. Una longitud de onda dual Shimadzu volando densitómetro lugar, el modelo CS-9301 PC (Tokio, Japón) se utilizó. Las condiciones eran las siguientes medidas: longitud de onda = 295 nm; modo de foto = reflexión, el modo de exploración zigzage =; ancho swing = 10.
El sistema LC (Agua, EE.UU.) consistió en una serie modelo 600 de la bomba LC y Modelo 600 unidad de control, un detector modelo 486 absorbancia ajustable, una válvula de inyección con 20 l de bucle constante, y un módulo de datos del modelo 746. columna Luna C18, 5 m, 25 cm × 4,6 mm (Phenomenex, CA, EE.UU.) se empleó. El FT-IR y MS se realizaron en el Centro de Micro Analítico, Universidad de El Cairo. espectros FT-IR se registraron en un espectrómetro Bruker Vector 22 (Bruker Instruments, Rheinstetten, Alemania) mediante pastillas de KBr en el rango (4,000-400 cm-1).

instrumento de espectrometría de masas, (Shimadzu, Japón), GC-MS-Qp-1000 espectrómetro EX cuádruples se utilizó. El sistema estaba operando en el régimen siguiente, la tensión de electrones: 70 eV, la temperatura de iones de la fuente: 200 ° C, el modo de ionización: IE, ACQ modo: exploración, velocidad de escaneo: 769.
2.2 Materiales y Reactivos

Rubro fue suministrada gentilmente por Memphis Co. (El Cairo, Egipto). Su pureza se ha certificado para contener 99,70%. cápsula Agreal fue fabricado por Memphis Co. bajo licencia de Laboratorios Synthelabo, Francia y etiquetados para contener 100 mg / cápsula. Todos los productos químicos utilizados fueron de grado analítico o LC. El agua para la CL ha sido elaborado por destilación de vidrio doble y filteration a través de una membrana de 0,47 micras filtro (Alltech Asociado EE.UU.,). acetato de amonio, 1 mM (Surechem Co., Reino Unido) se utilizó. El metanol (Lab. exploración, Irlanda) fue de grado LC. Ninhidrina (Sigma, EE.UU.), el ácido cloroformo, ácido clorhídrico (Fischer Scientific, Reino Unido), tolueno, amoniaco, ácido acético glacial, el peróxido de hidrógeno e hidróxido de sodio (Adwic Co., Egipto) fueron de grado analítico.

2.3 Condiciones cromatográficas
2.3.1 TLC
La placa se ha desarrollado en tolueno, metanol, amoniaco, cloroformo-10 5:3:6:0.1% (v / v) como un sistema en desarrollo. Durante 10 l detección y cuantificación de cada uno de la solución de muestra y las soluciones estándar se aplica. Las placas se desarrollaron de la forma habitual ascendente, secado al aire, y escaneado a 295 nm.


2.3.2 LC
La fase móvil fue metanol-acetato de amonio 1 mM en una proporción de 80:20 (v / v) y se ajustó a pH 3 con ácido acético. El flujo fue de 1 mL min-1. Todas las determinaciones se realizaron a temperatura ambiente, el volumen de inyección fue de 20 mL, y longitud de onda fue de 295 nm.

2.4 Elaboración de productos de degradación
Un pesado exactamente 50 mg de Ver. fue trasladado en tres matraces de fondo redondo. Los frascos fueron sometidos a alcalinos, ácidos y degradaciones de estrés oxidativo por reflujo con 50 mL de NaOH 1 M, 2 M de HCl y el 3% H2O 2 a 100 ° C durante 1 h. Luego, cada solución se neutralizó, se evaporó a cerca de 10 ml y cuantitativamente transferido a matraz aforado de 50 ml. El volumen se completa con agua destilada.

2.5 Las soluciones estándar y los gráficos de calibración
Las soluciones estándar se prepararon por dilución de la solución madre (1 mg mL-1) de la droga con metanol para TLC y la fase móvil para el método de LC. Los rangos de concentración fueron entre 100 y 1.000 mg mL-1 y de 1 a 100 mg mL-1 sobre las formas de TLC y LC, respectivamente. El área del pico promedio fueron graficados contra concentraciones.

Fig. Un LC cromatogramas de veraliprida y sus productos de degradación después de 0,5 horas, (a) 1 M NaOH, (b) 2 M HCl, (c) 3% H2O2, Fase móvil, 80:20 metanol-acetato de amonio (v / v), pH 3 a 25 ° C, K factor de retención, la selectividad α, R s resolución, T factor de cola.

3.3 Método de validación
Los métodos fueron validados con arreglo a normas ICH, documentando su linealidad, exactitud, precisión, límite de detección y cuantificación. Los resultados obtenidos fueron comparados estadísticamente con los métodos reportados y no hay diferencias significativas de la sustancia de drogas y medicamento. relaciones señal-ruido de 3 y 10 se utilizaron como criterios para la determinación del límite de detección y límite de cuantificación. La especificidad se evaluó, y el porcentaje de recuperación se calculó. La solidez fue investigado por la evaluación de la influencia de un pequeño cambio de las condiciones experimentales, tales como la resistencia orgánica, pH y caudal. Los resultados indican la capacidad del método para no ser afectada por pequeños cambios en las condiciones de los parámetros. prueba de aptitud del sistema se calculó y los resultados se muestran en la figura. 1.

3.4 Análisis de la formulación farmacéutica
Los métodos propuestos se han aplicado con éxito para la determinación de la droga en su formulación farmacéutica.

3.5 Cinética de Investigación

El proceso de degradación seguido pseudo cinética de primer orden. La tasa aparente de primer orden de la degradación constante y la vida media a cada temperatura se calcularon. Trazado de registro de valores obs K frente a 1 / T, el gráfico de Arrhenius fue encontrado para ser lineal. La energía de activación se encontró que 13,1 kcal mol-1. El valor sugerido la inestabilidad intermedio de la droga.

4 Conclusión
Los métodos sugeridos sólo se puede aplicar para estudios de estabilidad acelerada para predecir las fechas de caducidad de los productos farmacéuticos, y podría ser utilizado para las pruebas de pureza, en el control de calidad de los medicamentos estudiados.

References
1.Martindale (2007) The complete drug reference, 35th edn. The pharmaceutical press, London, 1736

2.Michele R, Antonella A (2005) J Neuropsychiatry Clin Neurosci. 17:252–253, PMID 15939983

3.Vincenzo DL, Giuseppe M, Maria CM, Elisa F, Ada D, Felice P (2006) Expert Opin Drug Saf 5:695–701. doi:10.1517/14740338.5.5.695


4.The Merck Index (2006) 14th edn. Merck and Co. Inc., Whitehouse Station, pp 1710

5.Staveris S, Jung L, Gamet G, Koffel JC (1985) J Chromatogr 338:79–88


6.ICH Q2A (1994) Validation of analytical methods: definitions and terminology. In: IFPMA (ed) International Conference on Harmonization, Geneva

7.ICH Q2B (1996) Validation of analytical procedure: method logy. In: IFPMA (ed) International Conferences on Harmonization, Geneva

8.Clark’s (2004) Analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem material, third edition. Pharmaceutical Press, London, p 293
......
Limited Short Communication
Validated Stability-Indicating Chromatographic Methods for the Determination of Veralipride in Presence of Its Degradation Products
Maissa Yacoub Salem1, Nahla Nour El Din Salama2, Lobna Mohammed Abd El Halim2 and Laila El-Sayed Abdel Fattah1

(1) Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
(2) National Organization for Drug Control and Research (NODCAR), 6 Abou Hazem Street, Pyramids, P.O. Box 29, Cairo, Egypt

Two sensitive and selective chromatographic methods were developed and validated for determination of veralipride in presence of its degradation products. Forced degradation studies were performed, using HCl, NaOH and 3% H2O2. The first method is based on thin-layer chromatographic separation of the intact drug spot from its degradation, followed by densitometric measurements. The second method is based on isocratic liquid chromatographic separation of the studied drug from its degradation on a reversed phase C18 column. The proposed LC method was utilized to investigate the kinetics of alkaline degradation process of the selected drug at different temperatures.
Keywords Thin layer chromatography - Column liquid chromatography - Stability studies - Veralipride


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1 Introduction
Veralipride (Ver) is an anti-psychotic drug, used in treatment of cardiovascular and psychological symptoms associated with the menopause [1–3]. It is N-[(1-allyl-2-pyrrolidinyl)methyl]-5-sulphamoyl-2-veretramide [4].

In literature, gas chromatography with mass spectrometry or flame ionization detector was used [5]. The International Conference on Harmonization (ICH) guidelines recommend that stress testing should be carried out to elucidate the inherent stability characteristics of the active substance. Acidic, alkaline and oxidative stability are required [6, 7]. Since no methods have been published for determination of Ver in presence of its degradation products, the aim of this work was to develop densitometric TLC and LC methods for determination of Ver in presence of its degradation products (Table 1). Furthermore, the developed LC method was used to investigate the kinetics of the alkaline degradation process.
Table 1 Results of assay validation obtained by applying the proposed TLC and LC methods for the determination of veralipride in drug substance and drug product
Parameters
Veralipride

TLC
LC

Linearity range
1–10 μg spot−1
1–100 μg mL−1

Accuracya

Mean ± RSD%b
99.83 ± 1.44
100.35 ± 1.45

Mean ± RSD%c
99.18 ± 0.676
99.58 ± 1.17

Precision

Intraday RSD%d
0.76
1.52

Interday RSD%d
0.49
0.54

LOD
0.041 μg spot−1
0.015 μg mL−1

LOQ
0.086 μg spot−1
0.044 μg mL−1

Specificity

Mean ± RSD%
100.39 ± 1.10
99.31 ± 1.08

Regression equatione

Slope
1.73 × 102
12.5 × 103

SE of the slope
2.55
83.21

Confidence limit of slopef
1.66–1.80 × 102
12.39–12.79 × 103

Intercept
9.29 × 102
11.80 × 103

SE of the intercept
15.49
4.57 × 103

Confidence limit of interceptf
8.86–9.72 × 102
10.55–22.67 × 103

Correlation coefficient (r)
0.999
0.999


a n = 6
bDrug substance
cDrug product
d n = 9
e Y = a + bc, where C is the concentration in μg spot−1 for TLC and in μg mL−1 for LC and Y is the peak area
f95% confidence limit

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2 Experimental
2.1 Instrumentation
TLC plates (20 × 20 cm2, aluminum plates precoated with 0.2 mm silica gel F254), were purchased from Macherey Nagel (Germany) and the samples were applied to the plates with a 25 μL Hamilton micro-syringe. A Shimadzu dual wavelength flying spot densitometer, Model CS-9301 PC (Tokyo, Japan) was used. The measurements conditions were as follows: wavelength = 295 nm; photo mode = reflection; scan mode = zigzage; swing width = 10.

The LC system (Waters, USA) consisted of a model series 600 LC pump and Model 600 controller unit, a Model 486 tunable absorbance detector, an injector valve with 20 μL constant loop, and a Model 746 data module. Luna C18 column, 5 μm, 25 cm × 4.6 mm (Phenomenex, CA, USA) was used. The FT-IR and MS were performed in the Micro Analytical Center, Cairo University. FT-IR spectra were recorded on a Bruker Vector 22 spectrometer (Bruker Instruments, Rheinstetten, Germany) using KBr pellets in the range (4,000–400 cm−1).

Mass spectrometry instrument, (Shimadzu, Japan) GC-MS-Qp-1000 EX quadruple spectrometer was used. The system was operating in the following regime, Electron voltage: 70 eV, Ion source temperature: 200 °C, Ionization mode: EI, ACQ Mode : scan, Scan speed: 769.

2.2 Materials and Reagents
Ver was kindly supplied by Memphis Co. (Cairo, Egypt). Its purity was certified to contain 99.70%. Agreal capsule was manufactured by Memphis Co. under license from Synthelabo Laboratories, France and labeled to contain 100 mg/capsule. All chemicals used were LC or analytical grade. The water for LC was prepared by double glass distillation and filteration through a 0.47 μm membrane filter (Alltech Associate, USA). Ammonium acetate, 1 mM (Surechem Co., UK) was used. Methanol (Lab. Scan, Ireland) was of LC grade. Ninhydrin (Sigma, USA), chloroform, hydrochloric acid (Fischer Scientific, UK) toluene, ammonia, glacial acetic acid, hydrogen peroxide and sodium hydroxide (Adwic Co., Egypt) were of analytical grade.

2.3 Chromatographic Conditions
2.3.1 TLC
The plate was developed in toluene–methanol–chloroform–10% ammonia 5:3:6:0.1 (v/v) as a developing system. For detection and quantification 10 μL each of the sample solution and standard solutions were applied. The plates were developed in the usual ascending way, air dried, and scanned at 295 nm.

2.3.2 LC
The mobile phase was methanol–1 mM ammonium acetate in a ratio of 80:20 (v/v) and adjusted to pH 3 with acetic acid. The flow rate was 1 mL min−1. All determinations were performed at ambient temperature, the injection volume was 20 μL, and wavelength was 295 nm.

2.4 Preparation of Degradation Products
An accurately weighed 50 mg of Ver was transferred into three round bottom flasks. The flasks were subjected to alkaline, acidic and oxidative stress degradations by refluxing with 50 mL 1 M NaOH, 2 M HCl and 3% H 2O 2 at 100 °C for 1 h. Then each solution was neutralized, evaporated to about 10 mL and quantitatively transferred to 50 mL volumetric flask. The volume was completed with distilled water.

2.5 Standard Solutions and Calibration Graphs
The standard solutions were prepared by diluting the stock solution (1 mg mL−1) of the drug with methanol for TLC and mobile phase for LC method. The concentration ranges were 100 to 1,000 μg mL−1 and 1 to 100 μg mL−1 for TLC and LC methods, respectively. The average peak areas were calculated and plotted versus concentrations.

2.6 Laboratory Prepared Mixtures
Laboratory prepared mixtures of Ver and its degradation products in the concentration range, 10–90% for TLC and 1–99% for LC were prepared and analyzed. The percentage recovery was calculated.

2.7 Application to Pharmaceutical Formulation
An accurately weighed amount of ten capsules equivalent to 100 mg of Ver was dissolved in 80 mL methanol and stirred with a magnetic stirrer for 30 min. The solution was transferred quantitatively to a 100 mL volumetric flask, diluted to volume with methanol, and filtered. Then the procedures were completed as mentioned above.

2.8 Kinetic Investigation of Alkaline Degradation Products
An accurately weighed 50 mg of Ver was dissolved in 50 mL 1 M NaOH, and 5 mL was transferred into a series of stoppered test tubes, and placed in a thermostatic water bath at different temperatures (90, 80, 70, 60, 50 °C) for different time intervals. The content of each tube was neutralized and transferred to 50 mL volumetric flasks and the volume was completed with mobile phase. Five milliliter were transferred into 10 mL volumetric flasks and the volume was completed with mobile phase. Then LC was applied.


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3 Results and Discussion
3.1 TLC
The experimental conditions such as developing system composition, scan mode and wavelength were optimized. Complete resolution was achieved by using toluene–methanol–chloroform–10% ammonia in a ratio of 5:3:6:0.1(v/v). Two degradants spots namely 2,3-(dimethoxy-5-sulfamoyl-phenyl)acetic acid with R f 0.11 and C-(1-allyl-pyrrolidin-2-yl)methylamine with R f zero appeared under acidic or alkaline degradation but one degradate was obtained namely 2,3-dimethoxy-N-(1-oxiranyl methyl-pyrrolidin-2-yl methyl)-5-sulphamoyl-benzamide with R f 0.21 through oxidative degradation. The R f of Ver was 0.49.

Degradate with R f zero was detected by ninhydrin [8]. All degradants were identified by FT-IR and mass spectroscopy.

3.2 LC
A satisfactory separation was obtained with mobile phase consisting of methanol–1 mM ammonium acetate 80:20 (v/v), pH 3 adjusted with glacial acetic acid. The specificity of LC is illustrated in Fig. 1.

Fig. 1 LC chromatograms of veralipride and its degradants after 0.5 h, (a) 1 M NaOH, (b) 2 M HCl, (c) 3% H2O2, Mobile phase, methanol–ammonium acetate 80:20 (v/v), pH 3 at 25 °C, K Retention factor, α selectivity, R s resolution, T Tailing factor

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3.3 Method Validation
The methods were validated according to ICH regulations by documenting their linearity, accuracy, precision, limit of detection and quantification. The results obtained were statistically compared with the reported methods and there is no significant difference for the drug substance and drug product. Signal-to-noise ratios of 3 and 10 were used as the criteria for determination of LOD and LOQ. The specificity was assessed, and the percentage recovery was calculated. The robustness was investigated by evaluating the influence of small change of experimental conditions, such as organic strength, pH and flow rate. The results indicate the ability of the method to remain unaffected by small changes in parameter conditions. System suitability test was calculated and the results are shown in Fig. 1.

3.4 Analysis of Pharmaceutical Formulation
The proposed methods were successfully applied for determination of the drug in its pharmaceutical formulation.

3.5 Kinetics Investigation
The degradation process followed pseudo first-order kinetics. The apparent first-order degradation rate constant and the half-life at each temperature were calculated. Plotting log K obs values versus 1/T, the Arrhenius plot was found to be linear. The activation energy was found to be 13.1 kcal mol−1. The value suggested intermediate instability of the drug.


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4 Conclusion
The suggested methods can simply be applied for accelerated stability studies to predict expiry dates of pharmaceuticals, and could be used for purity testing, in quality control of the studied drug.



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References
1. Martindale (2007) The complete drug reference, 35th edn. The pharmaceutical press, London, 1736

2. Michele R, Antonella A (2005) J Neuropsychiatry Clin Neurosci. 17:252–253, PMID 15939983

3. Vincenzo DL, Giuseppe M, Maria CM, Elisa F, Ada D, Felice P (2006) Expert Opin Drug Saf 5:695–701. doi:10.1517/14740338.5.5.695


4. The Merck Index (2006) 14th edn. Merck and Co. Inc., Whitehouse Station, pp 1710

5. Staveris S, Jung L, Gamet G, Koffel JC (1985) J Chromatogr 338:79–88


6. ICH Q2A (1994) Validation of analytical methods: definitions and terminology. In: IFPMA (ed) International Conference on Harmonization, Geneva

7. ICH Q2B (1996) Validation of analytical procedure: methodology. In: IFPMA (ed) International Conferences on Harmonization, Geneva

8. Clark’s (2004) Analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem material, third edition. Pharmaceutical Press, London, p 293