Question

Briefly explain the principles behind the method of extraction (HPLC) to isolate paracetamol and the internal standard.

(assume that you used Paracetamol)

Answer 1

Paracetamol is among the most worldwide consumed pharmaceuticals. Although its occurrence in the environment is well documented, data about the presence of its metabolites and transformation products is very scarce. The present work describes the development of an analytical method for the simultaneous determination of paracetamol, its principal metabolite (paracetamol-glucuronide) and its main transformation product (p-aminophenol) based on solid phase extraction (SPE) and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). The method was applied to analysis of river waters, showing to be suitable to be used in routine analysis. Different SPE sorbents were compared and the use of two Oasis WAX cartridges in tandem proved to be the most adequate approach for sample clean up and pre-concentration. Under optimized conditions, limits of detection in the range 40-67ng/L were obtained, as well as mean recoveries between 60 and 110% with relative standard deviations (RSD) below 6%. Finally, the developed SPE-HPLC/DAD method was successfully applied to the analysis of the selected compounds in samples from seven rivers located in the north of Portugal. Nevertheless all the compounds were detected, it was the first time that paracetamol-glucuronide was found in river water at concentrations up to 3.57μg/L.Dauble distilled water, Paracetamol was used from USP reference standard, caffeine, analytical grade
KH2SO4, and HPLC grade acetonitrile were obtained from Merck chemicals.
Instrument and Chromatographic Condition
The method development was performed with a High pressure liquid chromatographic system
consisting of a waters model 2695 solvent delivery system, a waters model 2996 photodiode array detector. The
system was controlled and data analysis were performed with the empower software. The detector was set at
265nm and peak areas were integrated automatically by using empower software. Separation was carried out at
ambient temperature using XDB C18 column (4.5mm x 250 mm, 5µm).
Preparation of Buffer Solution (Mobile Phase –A)
The accurate weights 1.36 g of Potassium dihydrogen phosphate dissolves in 1000 ml of distilled water
and filtered through 0.45µ filter paper. Preparation of Standard and Sample Solutions
The standard stock solution of paracetamol (150 mg) and caffeine (7.5 mg) were accurately weighed
and transferred to 100 ml standard flask with dissolved in the initial concentration of mobile phase solution and
make up to volume with same diluent. Further 1 ml of standard stock solution diluted to 100 ml with diluent to
yield a solution with final concentration of 15(µg/ml) and 0.075(µg/ml) of paracetamol and caffeine
respectively

Specificity
The ability of an analytical method to unequivocally assess the analyte in the presence of other
components can be demonstrated by evaluating specificity.
Linearity
The linearity of the method was determined at six concentration levels ranging from 25 to 150% level
for paracetamol and caffeine. The graph constructed by plotting peak areas versus concentration of solution.
Precision
The precision of the method was evaluated by five times inject of mixture of paracetamol and caffeine
standard solution.Accuracy
Accuracy was studied by adding known concentration (100 % level) of paracetamol and caffeine to the
dilute level of placebo preparation and comparing the actual and measured concentrationsimits of Detection and Quantification
Limits of detection (LOD) were established at s signal to noise ratio (S/N) of 3. Limits of quantification
(LOQ) were established at a signal to noise ratio more than 10.

is the branch of science dealing with the study of interaction between Electromagnetic radiation and matter. It is a most powerful tool available for the study of atomic and molecular structure/s and is used in the analysis of wide range of samples. Optical spectroscopy includes the region on electromagnetic spectrum between 100 Å and 400 μm. The regions of electromagnetic spectrum are shown in t

Region Wavelength
Far (or vacuum)ultraviolet 10-200 nm
Near ultraviolet 200-400 nm
Visible 400-750 nm
Near infrared 0.75- 2.2 μm
Mid infrared 2.5-50 μm
Far infrared 50-1

2.Visible spectrophotometry is one of the most frequently employed technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation absorbed by a substance in solution. Instrument which measure the ratio, or function of ratio, of the intensity of two beams of light in the U.V-Visible region ar qualitative analysis, organic compounds can be identified by use of spectrophotometer, if any recorded data is available, and quantitative spectrophotometric analysis is used to ascertain the quantity of molecular species absorbing the radiation. Spectrophotometric technique is simple, rapid, moderately specific and applicable to small quantities of compounds. The fundamental law that governs the quantitative spectrophotometric analysis is the Beer -Lambert law.