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Acetylin 500

Aspirin 500

Aspirin junior 100

Aspro 320

Ass 500 Dolormin 500

Acetylin 500

Aspirin 500

Aspirin junior 100

Aspro 320

Ass 500 Dolormin 500

ASS-Dura

Ch.-B. 074035 500

ASS-Fridetten

Ch.-B. 019026 500

Ch.-B. 020047 500

ASS-ratiopharm 500

ASS-Woelm 500

Temagin ASS 600

Ch.-B. 212142 600

Ch.-B. 212143 600

Trineral 600

According to predefined quality standards, the content of the active ingredient should be 95-105% of declaration and at least 80% of the compound should be released within 30 min under the conditions chosen.

The authors repeated this study 2 years later on 62 different aspirin preparations. They found that of the tested formulations, 20 (!) still did not meet the quality standards mentioned above [77]. Thus,

References

67 Buge, A. (1977) Zur Chemie der Salicylsaure und ihrer wichtigsten Derivate, in 100 Years of the Salicylic Acid as an Antirheumatic Drug, Vol. 42 (R34) (ed. H. Bekemeier), Martin-Luther-Universitäat HalleWittenberg, Wissenschaftliche Beitrage, pp. 14-38.

68 Wheatley, P.J. (1964) The crystal and molecular structure of aspirin. Journal ofChemical Society, 6036-6048.

69 Kim, Y. and Machida, K. (1986) Vibrational spectra, normal coordinates and infrared intensities ofaspirin crystal. Chemical & Pharmaceutical Bulletin, 34, 3087-3096.

70 Ouvrard, C. and Price, S.L. (2004) Toward crystal structure prediction for conformationally flexible molecules: the headaches illustrated by aspirin. Crystal Growth Design, 4, 1119-1127.

71 Bond, A.D., Boese, R. and Desiraju, G.R. (2007a) Onthe polymorphism of aspirin. Angewandte Chemie, International Edition, 46, 615-617.

72 Bond, A.D., Boese, R. and Desiraju, G.R. (2007b) On the polymorphism of aspirin: crystalline aspirin as intergrowth of two "polymorphic" domains. Angewandte Chemie, International Edition, 46, 618-622.

despite containing ASA as the active ingredient at identical amounts, not all aspirin preparations might be the same in terms of bioavailability of the compound.

The major functional consequence is the action of salicylate as protonophore, for example, in mitochondrial membranes, to uncouple oxidative phosphorylation because of the abolition of the membrane impermeability to protons (Section 2.2.3). Neither aspirin nor other salicylates exhibit comparable physico-chemical properties.

Aspirin is commercially available in many different galenic formulations. In vitro studies on bioequivalence provided different results for different formulations. However, the functional consequences for clinical use have not been studied sufficiently.

73 Whitehouse, M.W. (1964) Biochemical properties of antiinflammatory drugs. III. Uncoupling of oxidative phosphorylation in a connective tissue (cartilage) and liver mitochondria by salicylate analogues: relationship of structure to activity. Biochemical Pharmacology, 13, 319-336.

74 Horsch, W. (1979) Die Salicylate. Die Pharmazie, 34, 585-604.

75 Gordon, M.S., Ellis, D.J., Molony, B. et al. (1994) Invitro dissolution versus in vivo evaluation of four different aspirin products. Drug Development and Industrial Pharmacy, 20, 1711-1723.

76 Blume, H. and Siewert, M. (1986) Zur Qualitatsbeurteilung von acetylsalizylsaurehaltigen Fertigarzneimitteln. 1. Mitteilung: Vergleichende Reihenuntersuchung zur pharmazeutischen Qualitat handelsublicher ASS-Monopräparate. Pharmazeutische Zeitung, 47, 2953-2958.

77 Siewert, M. and Blume, H. (1988) Zur Qualitatsbeurteilung von acetylsalizylsaurehaltigen Fertigarzneimitteln. 2. Mitteilung: Untersuchung zur Chargenkonformität biopharmazeutischer Eigenschaften handelsublicher ASS-Präparate. Pharmazeutische Zeitung Wissenschaft, 131 , 21.

Summary

Aspirin and its main metabolite salicylic acid are poorly water soluble at acidic and neutral pH. The solubility increases markedly in alkaline pH and is more than 100-fold higher for the sodium salts as compared to the free acids. This is also valid for strong acidic pH.

Salicylates, in contrast to aspirin, have unique physicochemical properties. These are caused by the close steric neighborhood of the acetate hydroxyl group to the carboxyl group. This allows the formation of a chelate ring structure and facilitates the release of protons.

30 | 1 General Aspects 1.2.2

Determination of Salicylates

Measurement of salicylates in biological fluids, that is, mainly plasma and urine, is of interest for several purposes. The purpose also determines the selection ofthe method. Most frequent is the control of plasma levels to verify that the plasma concentrations are within the therapeutic range. Determination of plasma levels is also necessary in the case of intoxication and for controlling the efficacy of detoxification procedures. Plasma or urinary levels of salicylates allow checking patients' compliance, an important issue for long-term aspirin use in cardiovascular prophylaxis and a frequent explanation of the so-called aspirin resistance (Section 4.1.6). Finally, measurements are of interest to study the pharmacokinetics ofsalicylates in research, in particular, drug metabolism and interactions.

The therapeutic plasma levels of salicylate differ, depending on the indication. They are in the range of 100-200 mg/ml at anti-inflammatory doses and 50-100 mg/ml for analgetic purposes (see Figure 2.23). The plasma levels of unmetabolized ASA are approximately one order of magnitude lower. Thus, assay methods should be sensitive enough to detect amounts above 1 mg/ml [78]. In most cases, no separate determination of aspirin and salicylate is necessary because there is a rapid and complete conversion of aspirin into salicylate in vivo.

1.2.2.1 Gas-Liquid Chromatography

Gas-liquid chromatography (GLC) is the reference standard. The technique allows separate determination of ASA, salicylic acid, and their metabolites. The detection limit is 1 mg/ml.

1.2.2.2 High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is an alternative to GLC but more complex and time consuming. Reverse-phase HPLC techniques with photometric detection are the methods of choice [79]. This has the advantage that the complete spectrum of aspirin and its metabolites can be measured simultaneously. However, one major problem is associated with this type of assay. This is the spontaneous hydrolysis of aspirin to salicylate in protic solvents, including water, methanol, and plasma (Section 2.1.1). Thus, some degradation of aspirin may occur ex vivo. A modification of this technique for human plasma, including extraction of salicylates in organic solvents, allows simultaneous determination of aspirin and its metabolites down to the levels of 100 ng/ml with an interassay variation of less than 10% (Figure 1.10). This technique combines simplicity in sample treatment with stability of aspirin over several days (!) without significant decomposition [80].

1.2.2.3 Spectrophotometry

Spectrophotometry is the earliest and most widely used method for measuring serum salicylate levels. The classical assays are colorimetric assays, taking advantage from the intense red color of salicylate/ Fe3 + complexes. The technology is simple and particularly suitable for compliance measurements.

Trinder Method The Trinder method [81] is a colorimetric test where salicylic acid is determined by measuring the absorbance of the ferric ion-sa-licylate complex after total serum protein is precipitated by mercuric chloride and allowed to react with ferric iron supplied by ferric nitrate. This method involves the generation of a complex between salicylate and ferric ion. Amax of the ferric complex is 540 nm. Quantification is done by measuring light absorbance at this wavelength by a spectrophotometer.

TheTrindermethodis simple, inexpensive, rapid, and very reliable. Spectrophotometry lowers the detection limit to about 100 mg/ml. This is sufficient for therapeutic and toxic purposes. However, the Trinder method measures salicylate rather than ASA. (False) Positive results may be obtained with salicylamide or methylsalicylate. Conversely, the method can also be used to measure these compounds, for example, in the case of poisoning. The Trindertestisalsorathernonspecificandsensitiveto a large number of other acids and amines [82]. This also includes compounds and their metabolites, which are increased in patients with Reye-like symptoms because of hepatic (metabolic) failure [83]

before intake (control)

10 min after oral administration of aspirin500 mg 1 h after oral administration of aspirin500 mg

GA: Gentisic acid SUA: Salicyluric acid aspirin: Acetylsalicylic acid SA: Salicylic acid

MBA: 2-Methyl benzoic acid (internal standard)

GA: Gentisic acid SUA: Salicyluric acid aspirin: Acetylsalicylic acid SA: Salicylic acid

MBA: 2-Methyl benzoic acid (internal standard)

before intake (control)

10 min after oral administration of aspirin500 mg 1 h after oral administration of aspirin500 mg

Figure 1.10 Chromatograms of a standard mixture of acetylsalicylic acid and major metabolites (50 ng each) (a) and plasma levels of a volunteer before, 10 min after, and 1 h after oral administration of 500 mg aspirin (b) (modified after Ref. [80]).

(Section 3.3.3). More recently, Morris et al. [84] have described a two-step colorimetric method that, however, so far has only been used in the research.

Second-Derivative Synchronous Fluorescence Spec-

trometry Another method that allows simultaneous determination of ASA and its major metabolites in one assay is second-derivative synchro-

nous fluorescence spectrometry (SDSFS) [85]. This method appears to be the first nonchromatographic technology for the simultaneous determination of aspirin and its major metabolites in one singleserum sample. The technique is not sensitive to several other drugs, found frequently in the sera of healthy subjects (antipyrine, ibuprofen, indometh-acin, theophylline, and others).

Summary

Several methods are available to determine aspirin and its major metabolites in biological fluids, including plasma (serum), liquor, synovial fluid, and urine. Most of them have the necessary sensitivity (detection limit 1 mg/ml or less).

HPLC separation and subsequent identification of the spots by appropriate standards is the most frequentlyusedtechnology. Advantages are the simplicity and reproducibility of the method, a high sensitivity (detection limit about 100 ng/ ml), and the possibility of simultaneous deter-

mination of several aspirin metabolites together with aspirin itself in one sample. Disadvantages of this and some other technologies include the spontaneous (pH-dependent) and enzymatic hydrolysis ofaspirin. However, this problem can be solved by appropriate sample processing.

The Trinder method, a colorimetric assay, determines salicylate and is also useful and simple, though less sensitive. It exhibits a number of cross-reactions with other compounds, which might become relevant, for example, in hepatic failure. GC/MS is clearly the most reliable technology. However, it needs expensive equipment and experienced investigators.

32 | 1 General Aspects References

78 Svirbely, J. (1987) Salicylates. Methods in Clinical Chemistry (eds A.J. Pesce and L.A. Kaplan), C.V. Mosby Company, St. Louis, MO, pp. 417-424.

79 Klimes, J., Sochor, J., Zahradnicek, M. et al. (1992) Simultaneous high-performance liquid chromatographic determination of salicylates in whole blood, plasma and isolated erythrocytes. Journal of Chromatography, 584, 221-228.

80 Kees, F., Jehnich, D. and Grobecker, H. (1996) Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography. Journal of Chromatography B, 677, 172-177.

81 Trinder, P. (1954) Rapid determination of salicylate in biological fluids. The Biochemical Journal, 57, 301-303.

82 Kang, E.S., Todd, T.A., Capaci, M.T. et al. (1983) Measurement of true salicylate concentrations in serum from patients with Reye's syndrome. Clinical Chemistry, 29, 1012-1014.

83 Chu, A.B., Nerurkar, L.S., Witzel, N. et al. (1986) Reye's syndrome. Salicylate metabolism, viral antibody levels, and other factors in surviving patients and unaffected family members. American Journal of Diseases of Children, 140, 1009-1012.

84 Morris, M.C., Overton, P.D., Ramsay, J.R. et al. (1990) Development and validation ofan automated, enzymemediated, colorimetric assay of salicylate in serum. Clinical Chemistry, 36, 131-135.

85 Konstantianos, D.G. and Ioannou, P.C. (1992) Simultaneous determination ofacetylsalicylic acid and its major metabolites in human serum by second-derivative synchronous fluorescence spectrometry. Analyst, 117, 877-882.

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