Tartaric acid

Sugar and acid contents are major factors for the flavour of tomato fruits and high but balanced levels of sugars and organic acids are desired. Both, the sugar and acid contents are important traits for breeding [[1] (link), [2] , [3] , [4] ]. Fruits of cultivated tomato (Lycopersicon esculentum) contain mainly glucose and fructose and only trace amounts of sucrose, while wild tomato species, for instance Lycopersicon chmielewskii, may contain sucrose as a main sugar [5 (link)]. The contents of sugars and organic acids of tomato fruits are highly dependent on the developmental stage and ripeness [6 (link),7 ]. During ripening the total amount of sugars increases to approximately 4% with glucose being predominant in green, unripe fruits while red, fully ripe fruits contain typically slightly more fructose than glucose [5 (link),7 ]. With increasing maturity after ripening the sugar content declines again [6 (link)]. The content of organic acids is also developmentally controlled and has been reported to increase during ripening [8 ]. At all stages citric acid is the dominant organic acid but unripe green tomatoes may contain significant amounts of malic acid while its content in ripe fruits is fairly low [9 (link)]. Similar to sugars, citric acid declines with progressing maturation after ripening while the content of malic acid remains relatively constant [6 (link)].
Tomatoes, as climacteric fruits, can ripen off-the-vine and it is a common commercial practise to harvest mature green or breaker stage (incipient red colour) fruits and to ripen them in transit or destination [3 ]. However, fruits ripened off-the-vine were shown to contain less sugars but similar levels of organic acids compared to fruits ripened attached to the mother plant [10 ,11 (link)], a difference that may negatively impact the flavour. Due to the importance of sugar and organic acid contents of tomato for breeding, quality assessment and physiological investigations a number of methods have been developed for quantification of these compounds.
Sugars were traditionally analysed by their capacity to reduce copper (II) or silver(I) ions. However, these methods were labour and time consuming and allowed only a rough differentiation of sugars in reducing and non-reducing sugars. Nowadays, mainly chromatographic [12 ,13 (link)], electrophoretic [14 ,15 ] and enzymatic methods are used [6 (link),16 (link),17 (link)] but also NMR [11 (link)], FTIR [18 (link)] and NIR [19 ] are applied. A convenient method for analysis of sugars includes separation on an amino (NH2) column with acetonitrile/water mixtures as eluent and detection using a refractive index (RI) detector [12 ,13 (link)]. Separation is based on interaction of the NH2 groups of the stationary phase with hydroxy groups of the sugars. Roughly, the more hydroxy groups a sugar has the stronger it interacts with the stationary phase and the later it elutes. Consequently, monosaccharides elute first, followed by disaccharides and trisaccharides. In addition to the number, also the position of hydroxy groups on the molecule is crucial for retention, thus allowing separation of different mono-, di- and trisaccharides. This method has the advantage that the sample can be directly loaded, no derivatisation steps are required and that amino columns are comparably cheap. However, organic acids and other compounds present in samples may bind strongly or even irreversibly to the column, which may influence retention and separation of sugars and reduce column lifetime.
Organic acids are frequently analysed in fruits, juices and other types of biological fluids by reversed phase (RP) HPLC [[19] , [20] , [21] , [22] (link)], ion exclusion chromatography [12 ,23 ], gas chromatography [24 ,25 (link)], enzymatic assays [16 (link),17 (link)] and NMR spectroscopy [11 (link),26 (link)]. For RP-HPLC aqueous acidic buffers containing no or small amounts of organic modifiers are used as eluents. Detection is possible by UV absorption at 210 nm. Since the carboxyl group is a weak chromophore detection is not very sensitive but sufficient for detection of the main acids in fruits. However, a more serious problem is the extremely low selectivity of a UV detector operated at 210 nm. Compounds with conjugated double bonds, for instance phenolics and nucleotide phosphates, because of their strong UV absorption, show pronounced signals even at low concentrations. Such compounds may cause severe problems for quantification of some organic acids, particularly those with a low capacity factor like tartaric and malic acid. Efforts have been made to remove interfering compounds using custom-made anion exchange columns [12 ,27 ] but that requires handling of huge volumes and has thus not found broad application although promising results were obtained.
Here we use commercial solid NH2 solid phase extraction (SPE) columns for sample preparation. Under the conditions applied, sugars appear in the flow through while organic acids are well retained. Thus, the flow through is essentially free of organic acids and other compounds binding strongly to NH2 phases and can be used for quantification of sugars with amino columns and RI detection. The organic acids bound to the SPE columns are eluted with phosphoric acid and analysed by HPLC using a C18 column and detection by UV absorption at 210 nm. Including SPE enhances selectivity considerably since only acidic compounds are retained by the SPE column, while many UV absorbing compounds like phenolics are efficiently removed. It is also possible to elute the organic acids with trifluoroacetic acid, derivatise them by methylation and analyse the formed volatile methyl esters by GC–MS (see Supplementary Methods).
Lactose is added as internal standard for sugars and tricarballylic acid for organic acid to the samples. Both compounds are usually absent from tomato and other fruits. The use of internal standards compensates for losses during sample preparation and detector drift and renders precise volume control unnecessary except for pipetting of the sample, making the methods simple and highly reproducible.

The co-crystals were obtained by solvent-assisted mechanosynthesis as follows: the mixtures of a selected mole fraction of meloxicam and tartaric acid were mixed and ground for 5 to 10 min at room temperature using a mortar and pestle to obtain complete homogenization. After that, approximately 0.06 mL methanol was addedadded, and the grinding was continued until the complete evaporation of the solvent (Figure 1). After preparation, the mixtures were kept in a desiccator until they were studied.

L-tartaric acid 150.087 g/mol (LGC, Teddington, UK, batch G1057978, purity 97.9%), meloxicam 351.40 g/mol (batch 4198, purity 99.8%), methanol 32.04 g/mol (Merck, Darmstadt, Germany, purity 99.9%), sodium hydrogen phosphate (Merck, purity 99.8%), and sodium dihydrogen phosphate (Merck, purity 99.5%) were commercially available and used without further purification. All working solutions for the solubility tests were prepared using a deionized water/buffer solution of pH 7.4. To prepare 0.2 L of the phosphate-buffered solution, mix 19 mL of solution 2 M of sodium phosphate monobasic with 81 mL 2 M of sodium phosphate dibasic and bring to the mark with deionized water.