Diclofenac, detection

Among pharmacenticals, EE2 has been the snbject of particular recent attention becanse of its ability to canse endocrine disrnption in fish, as has been described in Chapter 15. Low levels of mixtnres of beta blockers, such as propranolol, metoprol, and nadolol have been detected in snrface waters, and there have been investigations of their possible effects on aqnatic invertebrates (Huggett et al. 2002). Veterinary medicines, too, have come nnder scrntiny for example, the dramatic effects of diclofenac on vnltnres, which will be discnssed shortly. Many questions remain to be answered abont the possible ecological effects of complex mixtures of pharmaceuticals and veterinary medicines. 

Photodegradation of the nonsteroidal anti-inflammatory drug diclofenac produced carbazole-l-acetate as the major product (Figure 1.12) (Moore et al. 1990). In a lake under natural conditions, it was rapidly decomposed photochemically though none of the products produced in laboratory experiments could be detected (Buser et al. 1998). 

Anti-inflammatory drugs such as Ibuprofen, acetaminophen, diclofenac, atorvastatin, and hydrochlorthiazide were detected in the highest concentrations in samples from three WWTPs in Spain [93]. These compounds were present in average concentrations from 43 to 117 ng/g, 42 to 103 ng/g, 28 to 75 ng/g, 21 to 65 ng/g, and 29 to 126 ng/g, respectively. 

For therapeutic drugs, the highest concentrations in the raw sludge corresponded to the analgesics diclofenac (209 ng g ) and ibuprofen (135 ng g-1), and the sulfonamide antibiotic sulfathiazole (143.0 ng g-1). Next in abundance were the diuretic compounds furosemide (79.9 ng g-1) and hydrochlorothiazide (41.3 ng g-1), and the analgesic ketoprofen (42.4 ng g-1). The remaining PhC were found at concentrations below 40 ng g The list of the 24 detected... 

Conventional WWTPs are, therefore, unable to remove wide ranges of pharmaceuticals and other compounds. For pharmaceuticals, although acute toxicity of aquatic organisms or chronic effects are unlikely with the present concentrations due to dilution effects, a wide range of pharmaceuticals are detected in the Ebro, and the overall toxicity of mixed pharmaceuticals may be high. Further studies are therefore required to assess the interactions of different compounds and the consequential health effects. In a similar manner to other pollutants, pharmaceuticals have a clear sensitivity to climate change through dilution effects, and the projected future decrease in annual precipitation could cause certain compound concentrations (e.g. anti-inflammatory diclofenac and p-blocker pranolol) to reach levels which may cause chronic effects [76]. 

Reverse osmosis (RO) removes greater than 90% of many compounds, and in numerous cases, RO is able to reduce their concentration below their detection limit. Nonetheless, lower removal rates have been noted in the case of diclofenac (55.2-60%) and ketoprofen (64.3%) [79]. 

Diphenhydramine, diltiazem, carbamazepine and norfluoxetine have been reported simultaneously in the same wild fish [107]. Moreover, diclofenac was found accumulating in vultures [119], fluoxetine, sertraline and the SSRl metabolites norfluoxetine and desmethylsertraUne were detected in fish [120]. Diclofenac bioaccumulation factors were 10-2,700 in the liver of fish and 5-1,000 in the kidney, depending on exposure concentrations [40, 121]. Gemfibrozil occurred in blood plasma of goldfish after exposure over 14 days at 113 times higher levels than in water [40]. 

Diclofenac sodium, famotidine and ketorolac were analysed utilising their formation of a coloured charge transfer complex with 2,4 dichloro-6-nitrophenol. The complexes were detected by UV/visible spectrophotometry at 450 nm. The method was not affected by the presence of common excipients in the formulations analysed. The precision and accuracy of the method was comparable to that of HPLC methods used to analyse the same samples. ... 

Diclofenac has been identified as one of the major pharmaceuticals in the waters in Berlin (Germany) at median concentrations of 0.47 pgL (Ternes et al., 2002 Ferrari et al., 2003). Other analgesics such as naproxen, phenazone, codeine, indome-tacin, fenoprofen, ketoprofen, phenylbutazone, and propyphenazone have also been detected in sewage and surface water (Heberer and Adam, 2005 Boyd et al., 2005), although data about their occurrence in the environment are not extensive. The data presented in Figure 2.5 are not exhaustive but are rather intended to show the great diversity in concentrations of some of the key PPCPs in wastewater. 

Despite the resolving power of TLC-MS-MS, few applications in drug residue analysis have been reported. One application concerns the HPTLC-MS-MS analysis of a number of nonsteroidal anti-inflammatory drugs, including salicylic acid and its glycine conjugate salicylhippuric acid, diclofenac, indomethacin, naproxen, phenacetin, and ibuprofen (67). Another application describes the detection and identification of some of these compounds or their metabolites in urine by TLC-MS-MS (67). 

Figure 6.9. Detection of diclofenac metabolite in rat bile by three LC-MS screening methods (a) full-scan BPC from LTQ-FTICR (b) MDF-treated full-scan BPC from LTQ-FTICR (c) ion current of NLS of 46 Da (d) ion cunent of PIS of m/z 250 (e) ion current of PIS of m/z 266.

Therefore, the product ion at m/z 250 and a loss of 46 Da were selected for the parent ion scan and the neutral loss scan, respectively. The NLS analysis detected both diclofenac and Ml along with several intense peaks from the bile matrix that were proven as false positives (Fig. 6.9c). The precursor ion scan of m/z 250 revealed the parent drug, but not Ml (Fig. 6.9d). A second PIS was conducted by following a specific product ion at m/z 266 (m/z 250 +oxygen), which was used to detect Ml (Fig. 6.9c). 

Corcoran, O., Nicholson, J. K., Lenz, E. M., Abou-Shakra, F., Castro-Perez, J., Sage, A. B., and Wilson, I. D. (2000). Directly coupled liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the identification of drug metabolites in urine Application to diclofenac using chlorine and sulfur detection. Rapid Commun. Mass Spectrom. 14 2377-2384. 

The relative bioavailability of different enteric diclofenac products was investigated with normal and artificially decreased gastric acidity [63], Only one generic product was found to be fully bioequivalent. Comparison with in vitro studies concluded that the general test on enteric-coated tablets according to Ph.Eur. did not detect any difference between the four products. A modified dissolution test without mechanical stress gave an indication on differences in the lag time of the different products. 

A highly sensitive and specific ELISA for the determination, in different types of water samples, of diclofenac, a commonly used nonsteroidal anti-inflammatory drug (NSAID), has been developed by Deng et al.357 This analyte belongs to the most frequently detected, pharmaceutically active compounds in the water cycle. The immunoassay was able to measure tap water samples directly— respective LOD and IC50 values were 6 and 60 ng E. On the other hand, surface water samples required fivefold dilution and the wastewater samples 10-fold dilution in buffer to be analyzed correctly the LODs were then 20 and 60 ng E, respectively. Recently, the development and validation of a highly sensitive and specific ELISA for the detection of pharmaceutical indomethacin in... 

Diclofenac sodium concentrations in aqueous solutions and in plasma were estimated by means of a published high performance liquid chromatographic technique ( 1 ). This method had a detection limit of 5 ng/ml and gave replicate values within 10% at levels above 20 ng/ml. Calibration curves were linear throughout the range of measured concentrations. 

Giachetti et al. [60] compared the performance of mass selective detector (MSD), electron capture detector (ECD) and nitrogen-phosphorus detector (NPD) of gas chromatography systems in the assay of six nonsteroidal antiinflammatory drugs in the plasma samples. As a practical test, six NSAIDs (mefenamic, flufenamic, meclofenamic and niflumic acids, diclofenac and clonixin) added to plasma samples were detected and quantified. The analyses were carried out after solvent extraction from an acidic medium and subsequent methylation. The linearity of response was tested for all the detection systems in the range of 1-25 ng/mL. Precision and accuracy were detected at 1, 5 and 10 ng/mL. The minimum quantifiable level for the six drugs was about 1 ng/mL with each of the three detection systems. 

Shinozuka et al. [91] developed a sensitive method for the determination of four anthranilic acid derivatives (diclofenac sodium, aluminium flufenamate, mefenamic and tolfenamic acids) by HPLC procedure. The four drugs were converted into methylphthalimide (MPI) derivatives in a constant yield by reaction with /V-chloromethylphthalimide at 60°C for 30 min. The production of the MPI derivatives were confirmed by mass spectrometry. The MPI derivatives of the four drugs were separated by HPLC using a C-18 bonded phase LiChrospher RP-18 column (250 x 4 mm i.d.) with acetonitrile-water (80 20, v/v) as mobile phase. The flow rate was 0.8 mL/min. The UV absorbance was measured at 282 nm. The calibration curves of the MPI derivatives of the drugs were linear from 1.0 to 5.0 pg/rnL. The detection limits of the four drugs were 0.5-5 ng. The extraction procedure for the four anthranilic acid derivatives added in the plasma and urine was performed by using Extrelut 1 column. Yields of column extraction of 100 pL of plasma and urine samples (containing 0.5 pg of anthranilic acid derivatives) with 6 mL of ethyl acetate were 84-106%. 

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