Concerted measures

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Quality control is one of a number of concerted measures that analytical chemists can take to ensure that the data produced in the laboratory are fit for their intended purpose.

In practice, fitness for purpose is determined by a comparison of the accuracy achieved in a laboratory at a given time with a required level of accuracy therefore it comprises the routine practical procedures that enable the analytical chemist to accept a result or group of results as fit for purpose, or reject the results and repeat the analysis.

So QC is an important determinant of the quality of analytical data, and is recognized as such by accreditation agencies.

Internal quality control is undertaken by the inclusion of particular reference materials, here called "control materials", into the analytical sequence and by duplicate analysis. The control materials should, be representative of the test materials under consideration in respect of matrix composition, the state of physical preparation and the concentration range of the analyte.

As the control materials are treated in exactly the same way as the test materials.

Quality control is a final check of the correct execution of all of the procedures that are prescribed in the analytical protocol and all of the other quality assurance measures that underlie good analytical practice. QC also required being as far as possible independent of the analytical protocol, especially the calibration, that it is designed to test.

Ideally both the control materials and those used to create the calibration should be traceable to appropriate certified reference materials or a recognized empirical reference method. When this is not possible, control materials should be traceable at least to a material of guaranteed purity or other well characterized material.

In a typical analytical situation several, similar test materials will be analysed together, and control materials will be included in the group. Often determinations will be duplicated by the analysis of separate test portions of the same material. Such a group of materials regarded as being analyzed under effectively constant conditions. The batches of reagents, the instrument settings, the analyst, and the laboratory environment will under ideal control as no unchanged occur during analysis.

Systematic errors should therefore remain constant during the experiment as the monitoring of these errors is of concern.

The control materials are the basic unit of QC which regarded as being carried out under repeatability conditions.

For example, reagents may degrade, instruments may drift, minor adjustments to instrumental settings may be called for, or the laboratory temperature may rise. However, these systematic effects are subsumed into the repeatability variations.

In few words; to achieve the definition of quality control which is set of procedures undertaken by laboratory staff for the continuous monitoring of operation and the results of measurements in order to decide whether results are reliable enough to be released, Issues specifically excluded the following:

  1. Quality control of sampling. While it is recognized that the quality of the analytical result can be no better than that of the sample, quality control of sampling is a separate subject and in many areas is not fully developed. Moreover, in many instances analytical laboratories have no control over sampling practice and quality.
  2. In-line analysis and continuous monitoring. In this style of analysis there is no possibly of repeating the measurement, so the concept of IQC as used in this document is inapplicable.
  3. Multivariate IQC. Multivariate methods in IQC are still the subject of research and cannot be regarded as sufficiently established for inclusion here. The current document regards multianalyte data as requiring a series of univariate QC tests. Caution is necessary in the interpretation of this type of data to avoid inappropriately frequent rejection of data.
  4. Statutory and contractual requirements.
  5. Quality assurance measures such as checks on instrumental stability before and during analysis, wavelength calibration, balance calibration, tests on resolution of chromatography columns, and problem diagnostics are not included. For present purposes they are regarded as part of the analytical protocol, and QC tests their effectiveness together with the other aspects of the methodology.


Ketoprofen150 mg



3-Benzoyl-α-methylbenzeneacetic acid


A white crystalline powder. M.p. 93° to 96°.

Slightly soluble in water, soluble in acetone, ethyl acetate, ethanol ,chloroform and ether.

Dissociation Constant:-PKa4.5.

Partition Coefficient:-LogP(octanol/buffer pH 7.4), 0.

Colour Tests.

Aromaticity (Method 2)—colourless/yellow; Koppanyi-Zwikker Test—violet.

Thin-layer Chromatography.

System TD—Rf 27; system TE—Rf 06; system TF—Rf 25; system TG—Rf 14; system TAD—Rf 41; system TAE—Rf 85; system TAJ—Rf 54; system TAK—Rf 82; system TAL—Rf 98. (Ludy Tenger reagent, orange.)

Gas Chromatography.

System GA—ketoprofen RI 2245; ketoprofen-Me RI 2090; system GD—Retention time of methyl derivative 1.45 relative to n-C16H34; system GL—ketoprofen-Me RI 2090; M(OH-)-Me2 RI 2250.

High Performance Liquid Chromatography.

System HAA—Retention time(s) 19.6min; system HD—k 2.4; system HV—Retention time 0.66 relative to meclofenamic acid; system HX—RI 495; system HZ—Retention time(s) 6.4min.

Ultraviolet Spectrum.

Aqueous acid—260 (A11=665a); aqueous alkali—262nm (A11=647a).

Infra-red Spectrum.

Principal peaks at wavenumbers 1656, 1693, 1284, 714, 690, 1226cm−1 (KBr disk). Two polymorphic forms may occur.

, 210, 103, 181.


Gas chromatography

In plasma: limit of detection 130μg/L, ECD—P.

Gas chromatography-mass spectrometry.

In plasma or synovial fluid: ketoprofen and ibuprofen, limit of detection for ketoprofen <2μg/L.

Disposition in the Body

Ketoprofen is readily absorbed after oral, rectal, or IM administration. About 75% of a single oral dose is excreted in the urine in 24h, mostly in the first 6h, about 90% of which is the glucuronide conjugate; hydroxylation may also occur.

Therapeutic concentration

After oral administration of 100mg as capsules, to 7 subjects, peak plasma concentrations of 6.0 to 14.3 (mean 10)mg/L were attained in 0.45 to 2.5h; a single rectal dose of 100mg produced peak plasma concentrations of 4.7 to 10.5 (mean 7.5)mg/L in 0.75 to 1.5h, and an IM dose of 100mg produced peak concentrations of 8.3 to 13.2 (mean 10.4)mg/L in 0.33 to 0.5 h. After oral doses of 50mg four times a day to 7 subjects, mean maximum steady-state concentrations of 5.6mg/L were reported.

After administration of a single oral dose of 200mg (as sustained-release granules) to 12 subjects, mean peak plasma ketoprofen levels of 4.51mg/L were attained in 2h and remained practically constant for at least 12h. The same dose of a conventional sustained-release capsule resulted in a mean peak concentration of 5.91mg/L at 4.17h and administration of 100mg ketoprofen, twice at a 12-h interval, as a prompt-release capsule produced mean peak plasma concentrations of 10.52mg/L 1.38h after the first dose and 12.80mg/L 1.46h after the second dose.

Half-life:-Plasma half-life, 1 to 4h.

Volume of distribution:-About 0.1 to 0.2L/kg.

Clearance:-Plasma clearance, about 1 to 2mL/min/kg.

Protein binding:-In plasma, about 95%.

Dose:-100 to 200mg daily.

Researches on Ketoprofen 150 mg:-

  1. Direct HPLC analysis of Ketoprofen in horse plasma applying an ADS-restricted access-phase.
  2. Chemical analysis applied to the radiation sterilization of solid Ketoprofen.
  3. Spectophotometeric determination of Ketoprofen and its application in pharmaceutical analysis.
  4. Production of R-(-) - Ketoprofen from an amide compound by Comamonas acidovorans KPO-2771-4.
  5. Evaluation of microcrystalline Chitosan properties as a drug carrier.
  6. Enantiospecific pharmacokinetic studies on Ketoprofen in tablet formulation using indirect Chiral HPLC analysis.

Summarization of full text

Direct HPLC analysis of Ketoprofen in horse plasma applying an ADS-restricted access-phase


Ketoprofen [2-(3-benzoylphenyl)-propionic acid] is a non-steroidal anti-inflammatory drug (NSAID) of the propionic acid class, which also includes pharmaceuticals such as ibuprofen, naproxen and fenoprofen. Ketoprofenis mainly used in human therapy in the treatment of arthritis because of its analgesic and anti-inflammatory properties. The FDA approved the use of ketoprofen in horses for the alleviation of inflammation and pain associated with musculoskeletal disorders. Published methods for the determination of plasma ketoprofen concentrations involve complex procedures such as liquid-liquid extraction and SFE manipulations. Initial work from our group involved the determination of ketoprofen in plasma using reversed-phase HPLC, injecting the residue of a diethyl ether extract of the acidified plasma samples, solubilized in the mobile phase. In order to avoid time-consuming manipulations, a simple, rapid and reproducible method using an automated column-switching liquid chromatographic system for the determination of ketoprofen applying UV detection was reported.

The direct and repetitive injection of untreated biological fluids into an HPLC setup and the subsequent analysis of low-molecular weight analytes are rendered possible by a column-switching setup and special pre column phases. The proposed method is based on the integrated sample clean-up configuration making use of the precolumn LiChrospher RP-18 ADS, 25 آ 4 mm, connected via the electrically driven six-port valve from a programmable autosampler to the narrow-bore reversed- phase analytical column Ecocart LiChrospher 125-3 filled with LiChrospher 5 mm 100 RP-18, where an on-line determination of ketoprofen is performed.

The use of RAM (restricted access material) phases is based on the complete non-adsorptive size-exclusion of macromolecules and on the simultaneous extraction of low-molecular weight analytes.



All solvents and chemicals used were of HPLC or analytical reagent grade and no further purification was carried out.

Ketoprofen was purchased from Sigma Chemical Company, phenacetin from Paris, France, monobasic potassium phosphate and acetonitrile (Lichro solv) were purchased from Merck.


The liquid chromatographic system used consisted of a pump, a programmable autosampler and a UV-vis detector .The HPLC parts were connected through an interface with a Compaq Deskpro XL 5133 2 GB computer for data handling.

Standard and sample preparation.

A standard solution of ketoprofen 400 mg/mL (high range) in phoshate buffer 0.1 M, pH7.0, was prepared, and dilutions were made to provide two working solutions of 40 mg/mL (medium range) and 4 mg/mL (low range).

The appropriate internal standard working solutions of phenacetin were prepared in phosphate buffer, 0.1 M, pH 7.0 with a concentration of 200 mg/mL (high range), 20 mg/mL (medium range) and 2 mg/mL (low range), respectively. All solutions were stored in dark glassware at about 8°C.

Blood samples (10.0 mL) were taken from the horse 5 min before drug administration and at 0, 2, 5, 10, 20 and 30 min, and at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after i.v. administration of 1.0 g ketoprofen. Blood samples were collected in vacuum tubes containing lithium heparin as anticoagulant. Plasma was separated by centrifugation at 2000g for 2 min and stored at ہ20C until analyzed.

Spiked plasma: the calibration standard solution was prepared by adding various volumes, respectively 10, 30, 50, 70 and 90 mL of the standard working solution in phosphate buffer, 0.1 M, pH 7.0, and made up to 100 mL with the latter, 100 mL appropriate working internal standard solution, 100 mL phosphate buffer, 0.6 M, pH 7.0, and 1000 mL drug-free plasma.

Unknown sample: 100 mL phosphate buffer, pH 7, 0.1 M, 100 mL appropriate working internal standard solution, 100 mL phosphate buffer, 0.6 M, pH 7.0, and 1000 mL plasma.

The prepared plasma solutions were filtered through a regenerated cellulose syringe filter and placed in 1.5 mL threaded vials provided with screw caps with a hole and slotted silicon/PTFE septa.

Chromatographic conditions.

Due to the dosing system of the auto sampler, different volumes (high range, 100 mL; medium range, 200 mL; and low range, 400 mL) can be injected in to the system with the standard syringe and brought onto the precolumn using the phosphate buffer, 0.1 M, pH 7.0, as the transporting solvent. After washing the ADS column with about 6.0 mL phosphate buffer to eliminate the plasma matrix, the precolumn was put in backflush mode with a mixture of phosphate buffer 0.05 M, pH 7.0: acetonitrile (80:20), applying a flow rate of 0.8 mL/min thus transporting the analytes on the reversed-phase column Ecocart 125-3 HPLC (cartridge) with a LiChrocart 4-4 guard column, both packed with LiChro- spher 5 mm 100 RP-18 leading to separation. The analytical column was placed in a water bath and kept at 35°C.

Table 1. Recovery of ketoprofen from spiked horse plasma


Concentration of Ketoprofen (ng/ml)

Recovery of ketoprofen(%)

Recoverycalculation ,ratio of Ketoprofen: phenacetin






















The UV detector was set at 260 nm; the retention times were respectively about 8.5 min for ketoprofen and 11.0 min for phenacetin. The precolumn was reconditioned with about 6 mL phosphate buffer, 0.1 M, pH 7.0.


LiChrospher ADS

LiChrospher RP-18 ADS has a pore size of approxi-mately 6 nm (physical diffusion barrier) and excludes macromolecules larger than 15 kDa in the void volume.Before HPLC analysis, macromolecular compounds have to be removed from the sample because of their precipitation by higher amounts of organic solvents and their binding on the surface of the packing material. At the outer surface of the spherical particles are bound hydrophilic, electroneutral diol groups, preventing inter- actions with the protein matrix.

The inner surface, covered by hydrophobic C-18 alkyl-chains, is freely accessible for low molecular weight analytes. Thus the packing material provides a direct extraction base, fully automated, on-column enrichment and subsequent analytical separation of low-molecular compounds from untreated plasma samples.

In LC-integrated sample preparation the sample is first fractionated into sample matrix and analytes by the use of the precolumn. This means that the protein matrix of a biological sample can be directly flushed into the waste, the analyte fraction meanwhile being selectively extracted and enriched on the stationary phase of the pre column.

Out of the three types of LiChrospher RP-ADS, covering the whole range of hydrophobic capacity factors, the most suitable precolumn for a given analyte has to be determined in each specific case.

The ADS RP-was omitted because of the small capacity factor of ketoprofen when eluting with 50 mM phosphate buffer pH 7.0. The retention of ketoprofen was shorter and the peak-form significantly better on the ADS RP-18 precolumn compared to the RP-8 phase. The system with the LiChrospher ADS PR-18 precolumn provided less disturbed chromatograms and more stable baselines.

Switching times

After developing a column-switching method, initial switching times have to be determined.

first the switching time for the fractionating step expressed in minutes or as a volume of washing liquid completing the sample preparation and coupling the precolumn to the analytical column, and second the

switching time for the transfer step. The fractionation step was considered complete when the detector signal reached the baseline. Depending on the injection volume, the time required for the sample preparation step may be adapted.

The complete elimination of matrix components has to be achieved in order to prevent interference with the subsequent separation of the analyte as well as to protect the analytical column. A guard-column for the latter is therefore strongly recommended. The precolumn lifetime amounts to about 80 mL of biological matrix when processing horse plasma.

The optimization of the transfer step consists of peak compression of the analytes eluting from the precolumn.

With reversed-phase columns, peak compression can be achieved by ensuring that the content of organic modifier in the mobile phase used for transfer and separation is higher than in the washing fluid. However, high organic modifier solvent contents may cause buffer precipitation, which can be the cause of clogging precolumns and tubings. To avoid protein precipitation, the concentration of the organic modifier, the pH and the ionic strength of the washing fluid applied for the sample loading must be non denaturating. As the run time of the analytical separation is about 15 min, the fractionation of the next sample can be performed simultaneously with the analyzing step of the preceeding sample analysis.

The overlap of sample preparation, analysis and reconditioning of the precolumn increases the overall sample throughput. However, ghost peaks or base-line abnormalities, originating from column-switching (eluent or pressure change) have to be considered to eliminate interferences with the analytical separation.

The HPLC system described was able to process about 40 horse plasma samples per 24 h. With precolumn equilibration during run analysis the sample throughput may even be increased up to 60 samples.


When adapting the time-consuming diethyl ether extraction of acidified plasma to the LC-integrated column switching technique, the recovery of the applied internal standard naproxen reached only 20%, probably due to its high protein binding properties, the recovery of ketoprofen already being satisfactory.

Increasing the molarity of the washing fluid from 0.05 to 0.1 M improved the recovery of naproxen to about 40%; further molarity increase was omitted in order to avoid precipitation in the mobile phase. As the mean value for the recovery of phenacetin from plasma, performed at three concentration levels, was 99.8% and as an acceptable separation was obtained, the latter compound was used as the internal standard.

The recoveries of ketoprofen from spiked samples at six different concentrations, were calculated by comparing the obtained peak areas with those from aqueous solutions.

A mean value of 96.7% was reached when peak areas of ketoprofen were used and of 96.8% when calculation was performed employing peak area ratios of ketoprofen/phenacetin.


The relationship was investigated between detector response and drug concentration in plasma samples spiked with known ketoprofen amounts, ranging from 40 to 40.000 ng/mL, in three different ranges each with the appropriate internal standard concentration .

Table2. Linearity of ketoprofen extracted from spiked horse plasma (performed on different days; n >or =3)


Concentration of Ketoprofen (ng/ml)

Injection volume(uL)

Linearity area of ketoprofen

Linearity ratio of area of

















Evaluation of the linear regression coefficient for each range and calculations based on ketoprofen peak areas and on peak area ratios ketoprofen/internal standard proved that an internal standard is not necessary. Moreover, the relationship between injection volume and peak areas, as expected, proved to be reliable.

Intra-day variations

Intra-day assay at three concentrations was performed on freshly prepared plasma samples. Calculation of the relative standard deviation performed on peak areas of ketoprofen, ranging from 0.3 to 0.8%, proved the variations to be acceptable. The values obtained with peak area ratios were slightly higher, from 0.4 to 1.2%.

Inter-day relative standard deviations were measured at six different ketoprofen-spiked plasma concentrations.

Table3. Inter-day determination of ketoprofen in horse plasma samples

Calculation of area

Calculation of ratio/area



RSD (%)



RSD (%)

























Limit of quantitation and limit of detection

The limit of quantitation, being the lowest concentration that can be quantified with acceptable accuracy, was 10 ng/mL. A ketoprofen concentration of 2 ng/mL plasma was considered as the limit of detection. The latter was calculated on the basis of three times the area of disturbing signals arising in the chromatogram with a capacity factor close to the k'-value of ketoprofen. These limits were established by a 400ہmL injection. The cited limits may be lowered by injecting larger volumes.


It is expected that the developed system may not only be applied to the determination of ketoprofen, but also to the assay of other drugs from various pharmaceutical groups.

In initial experiments the recoveries from plasma of some representatives of the barbiturate group, analgesics, local anesthetics and xanthines were controlled and proved to be satisfactory. Only the detector wavelength and the concentration of the organic modifier needed adaptation.

The essential features of the method are the novel precolumn packing LiChrospher ADS, with the advantage of direct and repetitive injection of untreated plasma samples, except for a filtration step. Moreover there is a potential for safer handling of possibly infectious biological fluids. An on-column enrichment of analytes with elimination of the protein matrix and a quantitave recoverage of ketoprofen is achieved.

Due to the quantitative elimination of the matrix, the application of an internal standard can be omitted. As the ADS column exhibits a long life-span there is a low cost per sample. A considerable reduction of the analysis times compared to manual methods for bioavailability studies is obtained together with an excellent linearity, good precision and accuracy.

A coupled-column system using ADS precolumn packings should have a broad application in pharmacokinetics, drug-monitoring and screening; further work in this area is in process.

Quality control is a process employed to ensure a certain level of quality in a product or service. It may include whatever actions a business deems necessary to provide for the control and verification of certain characteristics of a product or service. The basic goal of quality control is to ensure that the products, services, or processes provided meet specific requirements and are dependable, satisfactory, and fiscally sound. It is characterized by sensitivity and selectivity. In general, an increase in sensitivity results in a loss of selectivity. To get good results in chemical analysis, it is essential to correlate these two parameters.

HPLC method is widely used till now and the separation of the analytes is based on the differences in the analyte affinity for the stationary phase surface. It is accurate and precise method that can be used for determination of several pharmaceutical formulations (like capsules, sachets and herbal medicines) contains different combinations of several materials and in different concentrations.

Example of applications on HPLC:" Direct HPLC analysis of Ketoprofen in horse plasma applying an ADS-restricted access-phase.

Ketoprofen is an active constituent of Bio-profaned drug. It is used as anti-inflammatory drug. Ketoprofen helps relieve the inflammation and pain associated with rheumatoid arthritis, osteoarthritis, menstrual cramps or premenstrual pain and swelling. Ketoprofen is available in a non-prescription strength to treat minor aches and pains associated with the common cold, backache, muscular aches, toothache and menstrual pain. Its action release immediately and last for long period.