HPLC method for Lubiprostone

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

18 posts Page 1 of 2
Dear All,
I am developing a HPLC method for Lubiprostone but I am not getting Lubiprostone as a single peak. One small peak is there which only comes down to its half height and baseline stays there for about 1 min then main peak is coming. Main peak descends down to baseline. This way it is forming a bridge like structure between two peaks. Does anyone came across such problem or any body having a HPLC method for Lubiprostone?
I do not have any experience with this compound but generally the phenomena you describe occurs when you have two compounds in a relatively slow equilibrium. My uneducated guess is that you have anomer interconversion similarly to that seen when separating reducing sugars.

http://en.wikipedia.org/wiki/Reducing_sugar
Petrus Hemstrom
MerckSequant
Umea, Sweden
Bintang wrote:
I do not have any experience with this compound but generally the phenomena you describe occurs when you have two compounds in a relatively slow equilibrium. My uneducated guess is that you have anomer interconversion similarly to that seen when separating reducing sugars.

http://en.wikipedia.org/wiki/Reducing_sugar

Thanks Bintang
I think you are right. This compound may exist in two forms (Keto and hemiacetal forms). But I am not able to shift the equilibrium to either side. Use of Acid pH or Basic pH or Temperature is not helping. Could you share how did you manage anomer conversion in your case?
The way this is done with sugars is by using amines, either amino columns or as a mobile phase additive. You do not want to shift the equilibria just speed up the interconversion rate so it is much faster than the chromatographic time frame, that way you get only one peak.
Petrus Hemstrom
MerckSequant
Umea, Sweden
Bintang wrote:
The way this is done with sugars is by using amines, either amino columns or as a mobile phase additive. You do not want to shift the equilibria just speed up the interconversion rate so it is much faster than the chromatographic time frame, that way you get only one peak.

thanks Bintang today I will try this.
What is your sample diluent? What are your instrument conditions? Flow rate? Temperature?
Time flies like an arrow. Fruit flies like a banana.
bisnettrj2 wrote:
What is your sample diluent? What are your instrument conditions? Flow rate? Temperature?

Chromatographic conditions:
Column: Symmetry C18
Flow : 1.0mL/min
Mobile phase : Buffer(0.01M Kh2PO4, pH-3) : ACN (40:60)
Diluent : ACN (Also tried Buffer: ACN but same results)
Wavelength:210nm
I am also working on this compound. These two peaks are tautomers (keto and enol). I just add those two peaks to quantitate.

However, my sample concentration is very low, about 0.01 ug/mL. I use some fluorescent dye (PAMD) to labe it but the chromatogram gets very complicated. Do you have similiar problems?

Thanks.
I am doing a method for determination of related subtances in lubiprostone using 200nm as detection wavelength and i am not labeling it. Tell me if the signal is coming down to baseline between two peak(Keto and enol). I don't have any problem even if it apears as two peaks but the baseline between these two peaks is my concern. So far I am not able to reslove this issue. If anyone has good HPLC method please share with me.
The signal is not supposed to come down to the baseline. It's a dynamic transition between these two forms. There are indefinte number of molecules in these signal. The signal in between the two peak should be there.

By the way, what is your sample concentration? Do you have other exipient in your sample solution?
Sample concentration is 5mg/mL and i am working with pure Lubiprostone, no excipients. I am looking for trace level impurities in Lubiprostone.
Hola, este producto yo lo trabaje hace como un año revisando la validación del fabricante de la materia prima se observa este comportamiento en los cromatogramas debido a la presencia de los dos tautomeros, para mejorar esto pueden preparar una solucion mas diluida.

preparación de la muestra:
ejemplo: 10mg de lubiprostone en 50ml de diluyente (metanol 70%-Buffer pH4.5 30%) pueden otros buffer con pH entre 6 y 2 a concentraciones bajas entre 0.05mM - 0.025mM.

Condiciones cromatograficas:
Longitud de onda 294nm
Fase movil: Buffer fosfato monobasico de potasio 0.025M pH 4.5, 55% - Acetonitrilo 45%
Flujo: 1.5 ml/min
Temperatura de columna:25°C
Columna: ACE C18, 5mcm x 150mm x 4.6mm
Volumen de Inyeccion: 50mcl

Los tiempos de retencion para el tautomero-1 y el tautomero-2 de Lubiprostone son aproximadamente 5.8minutos y 9.5 minutos. tengo cromatogramas si necesitan ver los picos me dan un correo y se los envio.
what is the retention time of those 2 peaks?
when do they elute?
Hello
I think we need to use MS detector to get a reliable method
stevcorr wrote:
Hola, este producto yo lo trabaje hace como un año revisando la validación del fabricante de la materia prima se observa este comportamiento en los cromatogramas debido a la presencia de los dos tautomeros, para mejorar esto pueden preparar una solucion mas diluida.

preparación de la muestra:
ejemplo: 10mg de lubiprostone en 50ml de diluyente (metanol 70%-Buffer pH4.5 30%) pueden otros buffer con pH entre 6 y 2 a concentraciones bajas entre 0.05mM - 0.025mM.

Condiciones cromatograficas:
Longitud de onda 294nm
Fase movil: Buffer fosfato monobasico de potasio 0.025M pH 4.5, 55% - Acetonitrilo 45%
Flujo: 1.5 ml/min
Temperatura de columna:25°C
Columna: ACE C18, 5mcm x 150mm x 4.6mm
Volumen de Inyeccion: 50mcl

Los tiempos de retencion para el tautomero-1 y el tautomero-2 de Lubiprostone son aproximadamente 5.8minutos y 9.5 minutos. tengo cromatogramas si necesitan ver los picos me dan un correo y se los envio.


Hola, mi correo es ricardoa60@hotmail.com. Por favor envíame los cromatogramas, te lo agradezco!
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