(19) TZZ ¥_T

(11) EP 2 735 877 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 33/68 (2006.01) 08.03.2017 Bulletin 2017/10

(21) Application number: 13193944.9

(22) Date of filing: 21.11.2013

(54) A NOVEL NINHYDRIN REAGENT FOR ANALYSING NITROGEN-CONTAINING COMPOUNDS NEUARTIGES NINHYDRIN REAGENZ UND VERFAHREN ZUR VERWENDUNG DAVON NOUVEAU RÉACTIF NINHYDRINE ET SON PROCÉDÉ D’UTILISATION

(84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB WO-A1-2008/081959 CN-A- 102 618 621 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO GB-A- 1 349 491 SU-A1- 1 597 700 PL PT RO RS SE SI SK SM TR US-A- 3 778 230 US-A- 4 274 833

(30) Priority: 21.11.2012 GB 201220902 • R. BHUSHAN ET AL: "Direct thin layer chromatography enantioresolution of some (43) Date of publication of application: basic dl-amino acids using a pharmaceutical 28.05.2014 Bulletin 2014/22 industry waste as chiral impregnating reagent", JOURNAL OF PHARMACEUTICAL AND (73) Proprietor: JPP Chromatography Limited BIOMEDICAL ANALYSIS, vol. 21, no. 6, 1 January Plymouth, Devon PL9 0DZ (GB) 2000 (2000-01-01), pages 1143-1147, XP055099046, ISSN: 0731-7085, DOI: (72) Inventors: 10.1016/S0731-7085(99)00203-4 • Pitts, Leslie John • DATABASE FSTA [Online] INTERNATIONAL Ivybridge, Devon PL21 9BX (GB) FOOD INFORMATION SERVICE (IFIS), • Pallot, Michael Gerard FRANkFURT-MAIN, DE; 1971, NAKANISHI T ET Wembury, Devon PL9 0DZ (GB) AL: "Enzymic studies on cheese ripening. III. • Jones, Philip Rapid finger-printing on a mixed thin layer for Plymouth, Devon PL3 5BN (GB) qualitative analysis of casein hydrolysate. (translated)", XP002719439, Database accession (74) Representative: Akers, Noel James no. FS-1972-07-P-1107 N.J. Akers & Co 63 Lemon Street Truro, Cornwall TR1 2PN (GB)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 735 877 B1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 735 877 B1 2

Description determined by the intensity of the coloured product de- tected in the photometer by way of absorbance at the [0001] The present invention relates to a novel reagent specified wavelength. Accordingly, this method may be for analysing nitrogen-containing compounds, for exam- used qualitatively and quantitatively to determine which ple amino acids and the like, and to a method for using 5 amino acids are present in a test sample and the relative the same. concentrations of each. [0002] Amino acids are the components from which [0005] The colour reaction between ninhydrin and the proteins are formed, which in turn play a key role in many amino acid or amine is very slow at room temperature. biological processes. In some cases the presence or ab- It is significantly faster at elevated temperatures, but still sence of a particular amino acid in an individual can se- 10 takes many minutes, even at a temperature of 130°C and riously affect their health. For example, an individual suf- above. To maintain good chromatographic performance fering from the genetic metabolic disorder phenylketonu- the colour reaction needs to take place in a time period rea cannot metabolise phenylalanine; the accumulation of around one minute or less. To achieve this, hydrindan- of which severely affects their brain development. Ac- tin, the reduced form of ninhydrin, was found to be re- cordingly, methods for detecting free amino acids or de- 15 quired for the ninhydrin reagent to be effective and pro- termining the amino acid compositions of proteins are vide an acceptable rate of reaction. There have been a vital for the proper diagnosis and management of diseas- number of suggested reasons or mechanisms for ex- es. Similarly, such methods are important for the analysis plaining the ability of hydrindantin to speed up the forma- of commercial drugs, food and foodstuffs, as well as pro- tion of the coloured products at elevated temperatures. tein and enzyme research and development. More gen- 20 One suggestion is that hydrindantin acts as a stabiliser erally, the detection and identification of nitrogen-con- for one of the reaction intermediates. As such it is con- taining compounds finds applications across a wide sidered as an accelerator not a catalyst. range of disciplines, including, agricultural, biochemical, [0006] The term ’ninhydrin reagent’ refers to a solution clinical, environmental, food, forensic, histochemical, or solutions containing all of the constituents necessary microbiological, medical, nutritional, plant and protein 25 for use in the amino acid analysers. Accordingly, a nin- sciences. hydrin reagent comprises hydrindantin, ninhydrin and the [0003] At present, free or hydrolytically released amino requisite buffers and solvents. The ninhydrin reagent acids are typically detected using automatic amino acid may be formed by adding a separate solution of hydrin- analysers. In the 1950’s the first automated amino acid dantin to the ninhydrin solution. Although, it would be analysis method was developed by Moore, Stein and 30 preferable provide one solution comprising all of the con- Spackman, (Spackman DH, Stein WH, and Moore S. Au- stituents necessary for amino acid analysis, an unaccept- tomatic recording apparatus for use in the chromatogra- ably low shelf life of the ninhydrin reagent would result. phy of amino acids. Anal Chem, 1958, 30:1190-1206). Alternatively, hydrindantin may be produced in situ by This multi-stage process involves separating the amino adding a reducing agent to the ninhydrin solution, thereby acids by ion exchange liquid chromatography. A ninhy- 35 reducing a portion of the ninhydrin to hydrindantin. The drin reagent is pumped from a reagent reservoir, mixed formation of hydrindantin in situ occurs more or less in- with the eluent from the ion exchange column and passed stantaneously in the presence of a strong reducing agent. through a steel or plastic reaction coil, heated to the tem- However, both of these methods require the user to mix perature required for reaction. Ninhydrin reacts with all a separate solution of ninhydrin with a separate solution amino acids and related amine compounds to form highly 40 of hydrindantin or a suitable reducing agent before the coloured reaction products. Ruhemann’s purple is ninhydrin reagent is ready to be used in the amino acid formed by primary amines and primary amino acids and analysis. may be measured by absorbance of light at a wavelength [0007] In recent times it has become the preferred of 570nm. Other coloured reaction products, in particular practice for manufacturers to provide two bottles, one yellow reaction products, are formed by secondary45 comprising a solution of hydrindantin in an organic sol- amines and a number of secondary amino acids. These vent and the other comprising a solution of ninhydrin, an reaction products may be measured by its absorbance aqueous buffer and an additional organic solvent, both of light at a wavelength of 440nm. The coloured reaction bottles being tightly sealed under an inert gas atmos- products vary in intensity according to the concentration phere. The contents of the bottles are then mixed to form of amino acid. 50 the ninhydrin reagent shortly before or immediately prior [0004] The amino acid reaction products are passed to use in an amino acid analyser. through a photometer where the light absorbed by the [0008] Unfortunately, hydrindantin is a very difficult re- dye complexes is detected at suitable wavelengths, in agent to handle. It is particularly unstable in the presence particular 570nm and 440nm. The presence of different of air, the oxygen rapidly oxidising the hydrindantin back amino acids may be determined by chromatography. The 55 to ninhydrin. Only relatively small amounts of air are nec- identity of each amino acid is established on the basis of essary to seriously deplete the hydrindantin and thus its migration characteristics and thus its position on the substantially reduce the sensitivity of the colour produc- chromatogram. The concentration of the amino acid is tion. If air is not rigidly excluded from the reagent, the

2 3 EP 2 735 877 B1 4 hydrindantin concentration will slowly drop until no colour agents have been assessed for their suitability for use in reaction will take place in the time frame of the chroma- reagents necessary for amino acid analysis Reducing tographic analysis. agents for ninhydrin are preferably highly soluble in the [0009] Exposure of a ninhydrin reagent comprising hy- reagent solution, have an excellent reducing property drindantin to the surrounding air must therefore be kept 5 against ninhydrin, do not form coloured by products, are to an absolute minimum as even minute traces of oxygen inert to the equipment, and are stable and easy to handle. exposure will cause a steady loss in hydrindantin activity. However, as is discussed in more detail below, existing An inert atmosphere, usually nitrogen, may be used both reducing agents do not exhibit all of these characteristics in the preparation and during use of the ninhydrin rea- and therefore interfere to a significant degree with the gent. As can be appreciated, this requirement results in 10 sensitivity and accuracy of amino acid analysis in one or the need for complex equipment and handling proce- more ways. dures, to ensure hydrindantin activity does not deterio- [0014] During the continuing development of the nin- rate at an unacceptable rate before and/or during its use. hydrin reagent, a lot of attention has been paid to the use Notwithstanding these precautions being used, a ninhy- of strong reducing agents to produce hydrindantin in situ. drin reagent comprising hydrindantin will typically have 15 The original method by Moore, Stein and Spackman em- a shelf life of no longer than one month. ployed stannous chloride as the reducing agent. Howev- [0010] In addition, hydrindantin is insoluble in totally er, the amount of stannous chloride required to produce aqueous media. However, hydrindantin is soluble in a sufficient hydrindantin for a reasonably fast reaction number of organic solvents. Accordingly, organic sol- caused eventual precipitation of tin hydroxide com- vents are generally added in relatively high proportions 20 pounds, which in turn fouled and blocked the flow tubing. to the reagent to reduce the possibility of precipitation of The use of stannous chloride was therefore soon aban- hydrindantin during storage and use. Indeed, organic sol- doned and other reducing agents such as cyanide, ti- ventcontents up to as high as 75% by volume are typically tanous salts, borohydride and ascorbic acid were inves- used. The solvent may also comprise a combination of tigated. Cyanide could not be used commercially be- two or more different types of solvent, to ensure adequate 25 cause of toxicity issues and lacked the necessary stabil- dissolution of hydrindantin. Organic solvents used for this ity. More serious studies were carried out on sodium purpose typically include dimethylsulfoxide (DMSO), borohydride and ascorbic acid reducing agents. methylcellosolve, ethylene glycol and sulfolane. Yet still, [0015] For example, US 3,778,230 discloses a colour if insufficient organic solvent is used or the hydrindantin developing solution for use in automatic amino acid anal- concentration is too high, precipitation may occur on30 ysis by Liquid Chromatography, wherein ascorbic acid is standing or build up in the chromatographic apparatus, used as the reducing agent. In the method described, blocking the tubing. ascorbic acid is reacted with ninhydrin in the presence [0011] In light of the above mentioned difficulties as- of methyl cellosolve to form hydrindantin. Ascorbic acid sociated with using hyrdrindantin, alternative techniques was found to have several advantages, such as in- and equipment for amino acid analysis using typical nin- 35 creased solubility, a potent reducing property and sensi- hydrin reagents have been proposed. For example, tivity of colouration. In addition, ascorbic acid is readily US3,632,496 discloses a reagent generator formed from regenerated after deterioration compared with conven- an elongated housing having a channel through including tional reducing agents, such as stannous chloride. an inlet at one end for receiving a reagent and an outlet [0016] Despite these advantages, the ninhydrin rea- at the other for discharging activated reagent. The chan- 40 gent disclosed in US 3,778,230 comprising ascorbic acid nel is defined by surfaces of metallic material catalytically as reducing agent is vulnerable to oxidation. The ninhy- active for reducing ninhydrin to form the activated rea- drin reagent must first be prepared, typically in an inert gent. In this way, ninhydrin in its stable state may be container, at ambient temperatures. As a consequence, stored and activated in one vesicle. elaborateand time consuming procedures arenecessary [0012] US 4,359,323 relates to a liquid chromatograph 45 in order to minimise the risk of oxidation of hydrindantin. analytical system for amines. The system consists of a In addition, a considerable amount of time elapses in the chromatographic column, a reaction column and a loop method whilst separated amino acids are introduced to to recycle the mobile phase for reuse. The primary and the ninhydrin reagent before reaching the heated reac- most significant feature of this system is that the liquid tion coil, thus distorting the results recorded in the chro- mobile phase is comprised of a combination of an eluent 50 matogram. In addition, during this time, the ninhydrin re- and a substance which reacts with amines to produce a agent may be exposed to oxygen which has diffused into compound which can be detected photometrically. Ac- the analysis equipment. In fact US 3,778,230 admits that cordingly, this process only requires a single pump for the deterioration characteristics of the ninhydrin reagent complete operation. In spite of the reduced complexity comprising ascorbic acid were only equivalent to that of of the pumping system, the presence of hydrindantin in 55 Moore, Stein and Spackman comprising stannous chlo- the mixture will still give the same problems of instability ride (i.e. 15 days). Recovery of the colouring capacity and air sensitivity as with present commercial reagents. after deterioration due to air oxidation necessitates re- [0013] In addition to the above, a number of reducing addition of ascorbic acid. An analogous approach would

3 5 EP 2 735 877 B1 6 not have been possible using the conventional Moore, hydride is not easy to handle, nor is it particularly stable Stein and Spackman method, due to the low solubility of reducing agent. Reasonable stability is only achieved if stannous chloride. kept in non-aqueous solvents. The main advantage to [0017] Perhaps oneof the most notable disadvantages using sodium borohydride as reducing agent is that no to using ascorbic acid as a reducing agent is that it is 5 unwanted coloured products or precipitates are pro- known to form brown coloured decomposition matter up- duced. Two bottles during manufacture are still required on heating alone. US 3,778,230 suggests that the reduc- however, but the composition and means of mixing is tion products of reacting ascorbic acid with ninhydrin are rather different from the methods described above. One not brown coloured. However, there is a high risk that bottle contains sodium borohydride, ninhydrin and a non- use of ascorbic acid as reducing agent results in the for- 10 aqueous solvent. The other bottle contains an aqueous mation of coloured by-products as a result of thermal buffer and additional non-aqueous solvent. It can be as- decomposition of the ascorbic acid, thereby adversely sumed that the borohydride does not react with the nin- affecting the accuracy of the results recorded by the chro- hydrin until mixed with the aqueous reagent. matogram. [0023] Unlike theMoore, Stein and Spackman method, [0018] In a further example, US 4,274,833 discloses a 15 sodium borohydride and ninhydrin are mixed together in- ninhydrin reagent comprising a reducing agent (sodium line before the heated reaction chamber of the analyser. borohydride or stannous chloride) or hydrindantin itself, Accordingly, it has been asserted that the Hitachi reagent as well as a sulfolane water miscible organic solvent. may be used for up to a maximum of 12 months from Employing sulfolane as the solubilising agent is suggest- opening a sealed container of the reagent, rather than ed to have many advantages. In particular, sulfolane sol- 20 the standard 1 month. Nevertheless, borohydride is still ubilises hydrindantin and ninhydrin very well and does not fully stable and it is known that the presence of trace not react with any of the solution components. Accord- metals will accelerate its deterioration. As a result, a sig- ingly, the reagent is stable for long periods of time without nificant amount of the borohydride will typically be lost precipitation, known to result in the clogging of flow lines. before the end of one year. Although, in use, hydrindantin The lifetime of the ninhydrin reagent comprising hydrin- 25 is produced in-line, an extra pumping system or line is dantin, is claimed to be at least 3 months. However, as necessary for introducing sodium borohydride into the is the case with US 3,778,230, the ninhydrin reagent must analysis equipment. This increases the complexity and first be prepared,typically inan inert container, atambient cost of the system and the Hitachi reagent may not be temperatures. As discussed above, this requires elabo- usable on all instruments, unless they are fitted with such rate and time consuming procedures in order to minimise 30 an additional pumping system and supply lines. Further, the risk of oxidation of hydrindantin. in use, hydrindantin is formed from ninhydrin before con- [0019] Little has changed in the last 60 years since the tacting the eluted amino acids in the heated reaction discovery of ninhydrin based analysis requiring hydrin- chamber. dantin by Moore, Stein and Spackman. Rather, this meth- [0024] GB 1 349 941 concerns an early method for the od is still the most common technique used today. Al- 35 fluorimetric assay of primary amino groups that employs though this method has since been modified by using certain aryl alkyl aldehydes in combination with ninhydrin different reducing agents and/or solvents, disadvantages to give complexes that exhibit a high fluorescence. still include vulnerability to oxidation and low solubility in [0025] More recently, a method for detecting hetero- aqueous solutions as highlighted above. trophic bacteria in industrial circulating water is described [0020] Despite considerable research having been40 in CN 102618621 A. conducted with regard to finding alternative reducing [0026] It is clear that there remains a need for an im- agents, manufacturers have generally reverted to pro- proved ninhydrin reagent, which is less vulnerable to ox- viding separate solutions of hydrindantin and ninhydrin. idation and thus more easily handled before and during Typically, manufacturers provide two bottles, the first use and provides a longer shelf life. comprising a solution of hydrindantin in an organic sol- 45 [0027] Accordingly, in a first aspect, the present inven- vent and the second comprising a solution of ninhydrin, tion provides a ninhydrin reagent for use in the chroma- an aqueous buffer and additional organic solvent; both tographic analysis of amino acids, the ninhydrin reagent bottles being tightly sealed under an inert gas atmos- comprising: phere. The bottles are then mixed to form the ninhydrin reagent prior to use in an amino acid analyser. 50 ninhydrin; [0021] Most manufacturers claim a maximum shelf life an aqueous buffer, wherein the buffer maintains the of about 12 months before mixing and 2 to 3 months after pH at a value between 4.5 and 5.5; and a tempera- mixing. However, the life time of the mixed reagent is ture-dependent reducing agent, which agent is inac- even lower once attached to the analysis instrument and tive in the reduction of ninhydrin at a first tempera- can be significantly below 1 month. 55 ture, wherein the first temperature is up to 30°C, and [0022] Hitachi appears to be the only major manufac- active in reducing ninhydrin to hydrindantin at a sec- turer to provide a reducing agent for producing hydrin- ond temperature, wherein the second temperature dantin, in particular sodium borohydride. Sodium boro- is higher than the first temperature.

4 7 EP 2 735 877 B1 8

[0028] As noted above, the present invention provides vention comprises a temperature-dependent reducing a ninhydrin reagent for visualising nitrogen containing agent. A range of temperature-dependent reducing compounds, including but not limited to amino acids. The agents inactive in the reduction of ninhydrin at a first tem- ninhydrin reagent comprises a temperature-dependent perature have been found to be capable of reducing nin- reducing agent having a specific set of properties, as 5 hydrin to form hydrindantin at a second temperature, discussed in more detail below. As a result of this, unlike higher than the first temperature. known and/orcommercially available ninhydrin reagents, [0034] Unlike with commercially available ninhydrin re- the components of the ninhydrin reagent of the present agents therefore, the temperature-dependent reducing invention do not need to be stored separately and agents of the present invention are highly stable in the premixed just before use, and hence may be stored and 10 presence of oxygen at the first temperature. Accordingly, provided to a user in one bottle. In light of this, use of the elaborate and time consuming procedures are not re- ninhydrin reagent of the present invention will be less quired to circumvent deterioration of the reagents prior complex and less expensive than those which are cur- to use. Further, the formation and the rate of formation rently commercially available. of hydrindantin from ninhydrin can be controlled by var- [0029] The ninhydrin reagent of the present invention 15 ying the temperature. In this way, hydrindantin is pro- does not need to comprise hydrindantin and is preferably duced only when required, increasing the lifetime of the substantially free from hydrindantin. This is an advanta- ninhydrin reagent during use and reducing cost. geous aspect of the reagent of the present invention. [0035] A compound is suitable for use as the temper- [0030] The components of the reagent are stable at ature-dependent reducing agent of the ninhydrin reagent the first, lower temperature, at which they may be trans- 20 of the present invention, provided it is inactive in the re- ported and stored. Hydrindantin is formed when the com- duction of ninhydrin at the first temperature and active in ponents ofthe ninhydrin reagentare heated to the second reducing ninhydrin to hydrindantin at the second temper- temperature, at which temperature the reducing agent is ature, wherein the second temperature is higher than the active in the reduction of ninhydrin to hydrindantin. Ac- first temperature. In this respect, the term ’inactive’ when cordingly, the ninhydrin reagent is easily handled and 25 used in relation to the reduction of ninhydrin at the first controlled both before and during use on the amino acid temperature is a reference to the compound having an analysis equipment. In fact, the ninhydrin reagent of the activity at or below a maximum activity. Similarly, the term present invention may be stored and used very easily ’active’ when used in relation to the reduction of ninhydrin and efficiently in the presence of air for a considerable at the second temperature is a reference to the com- period of time. It is has been found that the ninhydrin30 pound having or exceeding a minimum activity, as dis- reagent of the present invention may have a shelf life cussed in more detail below. well in excess of 2 years. [0036] Compounds for use in forming the temperature- [0031] In light of the above, the ninhydrin reagent of dependent reducing agent of the ninhydrin reagent may the present invention may be used for applications other have a reducing activity capable of reducing ninhydrin at than amino acid analysis using an amino acid analyser 35 a rate comparable to those of known reducing agents. instrument. Due to its increased stability, the ninhydrin However, it is preferred that the temperature-dependent reagent may be used as a visualising agent in general, reducing agent of the ninhydrin reagent is less active and provided that the test sample comprises one or more reduces ninhydrin at a lower rate than those reducing organic nitrogen containing compounds. Whilst commer- agents employed commercially. This has the advantage cially available ninhydrin reagents may be used for pur- 40 of easier control of the reduction reaction. poses other than the detection of amino acids on amino [0037] In order to assess whether a particular com- acid analysers using ion exchange separation methods, pound has sufficient activity in the reduction of ninhydrin an improved ninhydrin reagent having the above men- at the second temperature, a first Protocol has been de- tioned characteristics may be more readily applied in a vised; details of which are provided in Example 1. Com- wide range of other analytical methods for detecting ni- 45 pounds meeting the requirements of Protocol 1 are con- trogen-containing organic compounds. sidered to have at least a minimum activity in the reduc- [0032] The use of the reagent of the present invention tion of ninhydrin to hydrindantin at an elevated or second is based on the well established amino acid and ninhydrin temperature. reaction. Accordingly, the coloured reaction products [0038] According to the method of Protocol 1, a com- may be subjected to photometry in known manner. In 50 pound is considered sufficiently active in reducing ninhy- particular, the relative absorbance sensitivities obtained drin if at a threshold temperature, that is the second tem- at the two most popular wavelengths of 570nm and perature, it has a minimum degree of activity, that is, is 440nm, are similar when using the present invention as able to reduce a given amount of ninhydrin in a specified presently achieved in amino acid analysers using com- period of time. In this protocol, the threshold temperature mercially available reagents. For many applications, only 55 is 100 °C. one monitoring wavelength may be required to determine [0039] According to the test protocol, ninhydrin and an primary and secondary amino acids. amino acid solution is mixed with a solution of the select- [0033] As noted above, the reagent of the present in- ed compound at a threshold temperature of 100 °C. After

5 9 EP 2 735 877 B1 10

20 minutes, the intensity of Ruhemann’s purple pro- Example 2. Protocol 2 may be performed prior to or after duced, if any, is calculated by measuring the absorption Protocol 1 provided in Example 1. More preferably, Pro- of the solution using a standard spectrophotometer at a tocol 2 is performed after Protocol 1 has been used to specified wavelength of 570 nm. A strong intensity of establish that the selected compound has the minimum colouration indicates that the selected compound has a 5 degree of activity required at the threshold or second high degree of activity at the threshold temperature. More temperature for the reduction of ninhydrin. preferably, for the purposes of assessing whether the [0044] According to the method of Protocol 2, a ninhy- selected compound has the minimum degree of activity drin reagent comprising the selected compound is pre- required for forming part of the ninhydrin reagent of the pared and stored at the first temperature in the presence present invention, a minimum absorbance of 0.3 is re- 10 of air. At set time intervals, a sample is taken from the quired. The minimum activity of the compound in the re- ninhydrin reagent, mixed with an amino acid solution and duction of ninhydrin at the second temperature is one heated to the second temperature. For the purposes of that provides an absorbance of at least 0.3 in the afore- continuity with respect to Protocol 1 provided in Example mentionedtest. An absorbance below about 0.3 indicates 1, the second temperature is 100 °C, as discussed above. that the compound is not sufficiently active at elevated 15 The intensity of Ruhemann’s purple produced, if any, is temperatures in the reduction of ninhydrin for use in the calculated by measuring the absorption spectra of the ninhydrin reagent of the present invention. More prefer- solution using a standard spectrophotometer. Measuring ably, the minimum absorbance to be achieved by the the intensity over successive samples, in particular de- candidate compound is at least 0.4. termining the rate of any decrease in intensity of colora- [0040] In addition to the above, compounds for use in 20 tion, indicates whether the long term stability of the se- forming the temperature-dependent reducing agent in lected compound is suitable for the compound to be used the ninhydrin reagent of the present invention must be in the ninhydrin reagent of the present invention. inactive in the reduction of ninhydrin at a first tempera- [0045] According to Protocol 2, a selected compound ture. is deemed not to be sufficiently stable at the first temper- [0041] The first temperature at which the compounds 25 ature and therefore not suitable for use in the ninhydrin are required to be inactive is lower than the second tem- reagent of the present invention, if the corrected absorb- perature. Typically, the first temperature is a temperature ance drops to less than 50% after 1 month. That is, com- or range of temperatures at which the reagent is typically pounds suitable for use in forming the temperature- de- stored and/or transported, prior to use in an amino acid pendent reducing agent in the ninhydrin reagent of the analyser or for the purposes of visualising nitrogen con- 30 present invention have an activity of at least 50% of their taining compounds in general. Existing ninhydrin rea- initial activity after 1 month. More preferably, the selected gents are typically stored at room temperature or below. compound is considered suitable for use in the ninhydrin Accordingly, the first temperature is preferably room tem- reagent of the present invention, if the corrected absorb- perature. As room temperature typically varies due to ance is no less than 50% after 3 months, still more pref- external factors, the first temperature is preferably up to 35 erably if the corrected absorbance is no less than 50% 30 °C, still more preferably up to 25 °C, more preferably after 6 months. Particularly preferred compounds are still at or below 20 °C. The first temperature is preferably those in which the corrected absorbance in Protocol 2 is greater than 0 °C, still more preferably greater than 5 °C, at least 50% after 12 months, still more preferably if the more preferably still greater than 10 °C. In one embodi- corrected absorbance is at least 50% of the initial ab- ment, the first temperature is from 5 °C to 25 °C. Yet still 40 sorbance after 24 months. more preferably the first temperature is from 10 °C to 20 [0046] It has been found that compounds having the °C. properties set out above and able to meet the require- [0042] The compounds identified as being inactive at ments of Protocol 1 of Example 1 and meet or exceed the first temperature include compounds which are not the requirements of Protocol 2 of Example 2 are most normally considered to have any significant reducing ac- 45 advantageous for use in a ninhydrin reagent. As noted tion at the first temperature but which become sufficiently above, using the methods provided in both Examples 1 strong reducing agents at the second temperature, re- and 2, a range of compounds have been found to be acting with ninhydrin to form hydrindantin. In addition, sufficiently stable at the first temperature whilst also hav- thetemperature-dependent reducing agents identifiedas ing the minimum degree of activity required at the second being inactive at the first temperature include compounds 50 temperature for use in the ninhydrin reagent of the known to have only weak reducing activity (that is below present invention. While these compounds may vary ac- the maximum activity defined above) at the first temper- cording to their degree of activity at the second temper- ature, but which become sufficiently strong reducing ature, they all have the minimum degree of activity re- agents at the second temperature, reacting with ninhy- quired for use in the ninhydrin reagent of the present drin to form hydrindantin. 55 invention. [0043] In order to determine whether a compound is [0047] As indicated, Protocol 1 of Example 1 and Pro- sufficiently inactive at the first temperature, a second Pro- tocol 2 of Example 2 have been used to screen a range tocol has been devised; details of which are provided in of compounds for their suitability for use as the temper-

6 11 EP 2 735 877 B1 12 ature-dependent reducing agent of the ninhydrin reagent Protocol 1 and 2. However, there are a significant number of the present invention. The ninhydrin reagent may com- of disaccharides and polysaccharides comprising a prise a single temperature-dependent reducing agent. hemiacetal or hemiketal group in their cyclic form. These Alternatively, the reagent may comprise a combination types of saccharides are capable of acting as tempera- of two or more temperature-dependent reducing agents. 5 ture-dependent reducing agents as defined by Protocol [0048] Suitable compounds for use as the tempera- 1 and 2. The disaccharides, lactose and maltose are two ture-dependent reducing agent of the ninhydrin reagent such examples. of the present invention are known in the art and are [0055] A non exhaustive list of disaccharides which commercially available have been found to be capable of forming the tempera- [0049] In one embodiment, the temperature-depend- 10 ture-dependent reducing agent of the present invention ent reducing agent comprises one or more saccharides. includes Trihalose, Cellobiose, Kojibiose, Nigerose, Iso- The saccharides may be conveniently grouped into mon- maltose, Sophorose, Laminaribiose, Gentiobiose, osaccharides, disaccharides and polysaccharides. Turanose, Maltulose, Palatinose, Gentiobiulose, Manno- [0050] Many saccharides have mild reducing proper- biose, Melibiose, Melibiulose, Rutinose and Xylobiose. ties and are sometimes called reducing sugars. A reduc- 15 [0056] Longer chain polysaccharides can be effective ing sugar can be defined as one containing a hemiacetal temperature-dependent reducing agents; the tetrasac- or hemiketal group in its cyclic form producing an alde- charose, stachyose, is one such example. Whilst much hyde or ketone group in its open ring form. All monosac- longer chain polysaccharides, such as starch are not suit- charides and many disaccharides are reducing sugars. able, partially hydrolysed starch such as the dextrins and [0051] Preferred monosaccharides include glucose 20 maltodextrins can be effective temperature-dependent and fructose, which in their cyclic forms have a hemia- reducing agents. It is to be appreciated that whilst long cetal and a hemiketal group respectively. Glucose and chain polysaccharides may be suitable temperature-de- fructose were both found to have sufficient reducing ac- pendent reducing agents, they are not preferred as they tivity at the second, threshold temperature whilst remain- can give more viscous solutions and increase the possi- ing stable at the first temperature in the presence of air, 25 bility of precipitation. when subjected to the aforementioned Protocol 1 and [0057] The temperature-dependent reducing agent Protocol 2. may also comprise one or more carboxylic acids and/or [0052] Fructose was found to have a shelf life of ap- their salts. In general, simple monofunctional carboxylic proximately 3 years at room temperature. Although not acids, comprising only a single carboxylic acid group and as reactive in the reduction of ninhydrin at the second 30 no other functional group, have not been found capable temperature, glucose was found to be more stable than of acting as temperature-dependent reducing agents ac- fructose, having a shelf life of longer than 4 years at room cording to Protocol 1 and 2. Such monofunctional car- temperature. Accordingly, if a longer shelf life is required, boxylic acids include acetic, propionic, and benzoic acid. the ninhydrin reagent of the present invention preferably However, formic acid and its salts have been found to be comprises glucose. 35 capable of acting as temperature-dependent reducing [0053] Other monosaccharides for use as the temper- agents according to Protocol 1 and 2. ature-dependent reducing agent of the ninhydrin reagent [0058] Suitable salts of carboxylic acids for use in the of the present invention include such compounds as glyc- temperature-dependent reducing agent include metal eraldehyde and galactose, which are both aldoses. A non salts, in particular Group I and Group II metal salts, for exhaustive list of monosaccharides which have been40 example potassium, sodium, calcium and magnesium found to be capable of forming the temperature-depend- salts. Sodium formate, the sodium salt of formic acid was ent reducing agent of the present invention includes the found to be sufficiently active in reducing ninhydrin to aldoses, Ribose, Xylose, Erythrose, Threose, Lyxose, hydrindantin at a second temperature and sufficiently in- Arabinose, Allose, Altrose, Mannose, Gulose, Idose, Ta- active in the reduction of ninhydrin at a first temperature lose and L-Glycero-D-manno-heptose and the ketoses 45 according to Protocol 1 and Protocol 2 respectively. In Dihydroxyacetone, Erythrulose, Ribulose, Xylulose, Psi- particular, sodium formate was found to have a reducing cose, Sorbose, Tagatose and Sedoheptalose. An aldose activity in-between that of glucose and fructose. Further, is a monosaccharide which has an aldehyde group in its the shelf life was similar to that of glucose having a shelf linear form and a ketose is one which has a ketone group life in excess of 4 years. in its linear form. 50 [0059] Other carboxylic acidsand/or their salts suitable [0054] In general, some disaccharides and polysac- for use as temperature-dependent reducing agents ac- charides will not have the properties required to form suit- cording to the present invention are those comprising at able temperature- dependent reducing agents as defined least one additional reducing group, for example one or by the test Protocol and batch stability test. For the dis- more of a hydroxy ketone or aldehyde group. Suitable accharides they can be reducing or non-reducing sugars. 55 salts of the carboxylic acids include metal salts, for ex- For example sucrose is a non-reducing disaccharide and ample salts of metals in Groups I or II of the Periodic it was found to be not sufficiently active in the reduction Table. Examples of suitable salts include potassium, so- of ninhydrin to hydrindantin when assessed according to dium, magnesium and calcium salts.

7 13 EP 2 735 877 B1 14

[0060] Preferred carboxylic acids have from 1 to 24 that at concentrations higher than 20% w/v or v/v, com- carbon atoms, more preferably 1 to 18 carbon atoms, still ponents of the reagent may be more likely to precipitate more preferably from 1 to 12 with C1 to C6 aldehydes out of solution or cause other components to precipitate and ketones being especially preferred. The compounds out of solution and accordingly concentrations below may be straight chained, branched or cyclic. 5 20% are to be preferred. [0061] Further, compounds comprising one or more al- [0066] When preparing the ninhydrin reagent for use dehyde and ketone groups, with or without the presence in an analytical method, the starting concentration of the of hydroxyl groups, (including the simple aldehydes and temperature-dependent reducing agent is preferably ketones themselves) have been found to meet the re- similar to that which was identified in Protocol 1 provided quirements of Protocols 1 and 2. Preferred compounds 10 according to Example 1, as responsible for providing an of this type have from 1 to 24 carbon atoms, more pref- actual absorbance of at least 0.3, more preferably an erably 1 to 18 carbon atoms, still more preferably from 1 absorbance from 0.3 to 0.7. More preferably, as a result to 12 with C1 to C6 aldehydes and ketones being espe- of repeating the test Protocol according to Example 1 cially preferred. The compounds may be straight several times, each time increasing the percentage con- chained, branched or cyclic. Examples of suitable alde- 15 centration of the temperature-dependent reducing agent, hydes and ketones include acetone. theconcentration which provides thehighest absorbance [0062] The temperature-dependent reducing agent between 0.6 and 0.7 without precipitating out of solution may also comprise one or more inorganic compounds, or adversely affecting the other components of the nin- provided they too meet the requirements of Protocol 1 hydrin reagent is used as the starting concentration. and 2. In particular, inorganic compounds comprising sul- 20 [0067] The ninhydrin reagent of the present invention phur in low oxidation states such as sulfite and thiosulfate further comprises ninhydrin and an aqueous buffer. Nin- and/or inorganic compounds comprising phosphorus hydrin and suitable aqueous buffers are both commer- oxyacids in low oxidation states such as phosphites and cially available and used in existing ninhydrin reagents. hypophosphites may be particularly suitable. For exam- [0068] The ninhydrin reagent of the present invention ple, it has been found that phosphorous acid, including 25 may comprise ninhydrin in any suitable amount. Prefer- its phosphite salts such as lithium, sodium and potassi- ably, the concentration of ninhydrin in the reagent is from um, passes both Protocol 1 and 2 with an activity between 0.5 to 3% w/v. More preferably, the concentration of nin- that of fructose and sodium formate. hydrin is from 1 to 2.5% w/v. Yet still more preferably, [0063] The temperature-dependent reducing agents and as Protocol 1 according to Example 1, the concen- for use in the ninhydrin reagent of the present invention 30 tration of ninhydrin in the reagent is about 2% w/v. aregenerally knownin theart and areeither commercially [0069] Any suitable aqueous buffer may be used in the available or may be prepared in a manner analogous to ninhydrin reagent. Suitable buffers are known in the art. known synthesis routes. An acidic buffer is preferred. More preferably, a weak [0064] It is to be appreciated that other compounds or acidic buffer is preferred so as to maintain the pH at a classes of compounds which have not been identified 35 value of between 3 to 7, more preferably from 4 to 6, still above may be suitable components for use as the tem- more preferably from 4.5 to 5.5. A particularly suitable perature-dependent reducing agent of the ninhydrin re- pH is about 5.2. agent of the present invention, provided they meet the [0070] The buffer may be prepared from any weak acid requirements of both Protocol 1 of Example 1 and Pro- and one of its conjugate bases/salts. The acid may be tocol 2 of Example 2. In general, in addition to meeting 40 an organic acid or an inorganic acid. Organic acids are the requirements of Protocol 1 and Protocol 2, the tem- preferred, examples of which include acetic acid, etha- perature-dependent reducing agent should be readily noic acid and propanoic acid. One preferred combination soluble in or miscible with water and not cause precipi- is acetic acid and one of its salts. The acid salt may be tation when combined with other components of the re- any suitable salt. The salt may be formed from any metal agent and/or the components used or produced in the 45 cation. However it is preferable that the salt is formed analysis methods. It is also advantageous that the tem- from any one of the alkali metals, in particular lithium, perature-dependent reducing agent is substantially non- sodium or potassium. toxic. [0071] As noted above, hydrindantin is insoluble in a [0065] In light of the above the ninhydrin reagent may totally aqueous media. In light of this, commercially avail- comprise the temperature-dependent reducing agent in 50 able ninhydrin reagents, comprising hydrindantin, cannot any suitable concentration. This will vary according to be manufactured or used without the presence of a sub- the temperature-dependent reducing agent employed, in stantial amount of organic solvent. It is an advantage of particular according to such factors as the degree of ac- the present invention however, that the ninhydrin reagent tivity at the second temperature. The concentration of need not comprise an organic solvent or mixture of or- the temperature-dependent reducing agents may be55 ganic solvents. As a consequence, the manufacturing from 0.01% to 75% w/v or v/v. More preferably, the con- costs are reduced, the reagent is less harmful to the en- centration of the temperature-dependent reducing vironment and waste disposal procedures are simplified. agents is from 0.01% to 20% w/v or v/v. It has been found [0072] The presence of an organic solvent in the nin-

8 15 EP 2 735 877 B1 16 hydrin reagent may affect the efficacy of the reagent. The be used as a visualising agent to identify and/or quantify sensitivity of the reagent of some embodiments of the a wide range of nitrogen-containing compounds. present invention that do not comprise an organic solvent [0078] In a preferred embodiment however, the ninhy- has been found to be approximately 30% lower than that drin reagent of the present invention is used for amino of commercially available reagents which comprise an 5 acid analysis, in particular using known and standard organicsolvent or mixture of organicsolvents. In addition, amino acid analysers. in the absence of organic solvents, there may be a greater [0079] Accordingly, in a second aspect, the present risk of precipitation of the resulting hydrindantin. invention provides for the use of a ninhydrin reagent in [0073] Notwithstanding the above, the reagent of the a method for analysing nitrogen-containing compounds, present invention, when formulated without an organic 10 in particular amino acids and the like, wherein the ninhy- solvent, provides at least acceptable results when used drin reagent comprises; for amino acid analysis. In addition, optimisation of the ninhydrin; concentration of the temperature- dependent reducing an aqueous buffer; and agent according to Protocol 1 and 2 will minimise the risk a temperature-dependent reducing agent, which agent of precipitation. 15 is inactive in the reduction of ninhydrin at a first temper- [0074] In an alternative and preferred embodiment, the ature and active in reducing ninhydrin to hydrindantin at ninhydrin reagent of the present invention further com- a second temperature, wherein the second, threshold prises an organic solvent or mixture of organic solvents. temperature is higher than the first temperature Organic solvents typically used in commercially available [0080] The ninhydrin reagent may be used in a method reagents include, dimethylsulfoxide, ethylene glycol, pro- 20 for analysing, for example identifying and/or quantifying, pylene glycol, sulfolane, hydroxy ethers such as carbitol, one or more nitrogen-containing compounds. The meth- propylene glycol monomethyl ether and methylcellosolve od is suitable for analysing a range of nitrogen-containing and simple alcohols such as methanol, either alone or compounds. In fact, as noted above the method may be as mixtures. The addition of an organic solvent will in- used for analysing both organic and inorganic nitrogen crease the sensitivity of the ninhydrin reagent of the25 containing compounds. present invention. This should be taken into account [0081] The ninhydrin reagent may be used in a method when using the preferred ninhydrin reagent on a chro- for analysing, for example identifying and/or quantifying, matographic system, as is discussed in more detail be- one or more nitrogen-containing compounds. The meth- low. od is suitable for analysing a range of nitrogen-containing [0075] The total concentration of organic solvent used 30 compounds. In fact, as noted above the method may be will vary according to the nature of the organic solvent. used for analysing both organic and inorganic nitrogen In particular, the total concentration of organic solvent containing compounds. used will vary from 10%v/v to 75%v/v. More preferably, [0082] The method of the present invention is particu- the total concentration of organic solvent used will vary larly suitable for analysing amino acids and compounds from 25%v/v to 65%v/v. Yet still more preferably, the total 35 formed from amino acids, such as proteins, peptides, and concentration of organic solvent used will vary from the like. The following description describes the analysis 35%v/v to 55%v/v. Whilst any of the above commercially of amino acids. It is to be understood that the other afore- available organic solvents may be used, in accordance mentioned nitrogen-containing compounds may be proc- with the chromatography examples shown below, the essed in an analogous manner. method of the present invention preferably employs40 [0083] The nitrogen-containing compounds may be 40%v/v to 55%v/v ethylene glycol. present in any liquid medium or stream. The method of [0076] The ninhydrin reagent of the present invention the present invention is particularly suitable to the anal- may be used in accordance with known and standard ysis of an eluent from the outlet of an ion exchange col- procedures and methods for the detection of nitrogen- umn containing one or more amino acids in an ion ex- containing compounds. The ninhydrin reagent used in 45 change liquid chromatography procedure. such methods is that which has been defined above in [0084] The method of the present invention may be the first aspect of the present invention. Accordingly, the carried out using a conventional automatic amino acid ninhydrin reagent comprises a temperature-dependent analyser. As noted above, automatic analysers typically reducing agent inactive at a first temperature and active comprise means for separating amino acids into their re- at a second threshold temperature to reduce ninhydrin 50 spective components, an input system for introducing a to form hydrindantin according to Protocol 1 and Protocol ninhydrin reagent intothe system, allowingthe separated 2 provided in Examples 1 and 2 respectively. amino acids and ninhydrin reagent to react at elevated [0077] As previously discussed, the ninhydrin reagent temperatures to form coloured substances and means of the present invention must be inactive at a first tem- for recording the absorbance of the coloured substances perature and active at a second temperature to reduce 55 at specified wavelengths. In this way, the amino acids ninhydrin to form hydrindantin, wherein the second tem- present within a test sample and the relative concentra- perature is higher than the first. Due to its increased sta- tions of each may be determined. bility, the ninhydrin reagent of the present invention may [0085] Amino acids are typically separated using tech-

9 17 EP 2 735 877 B1 18 niques which discriminate on the basis of size, solubility, from 15 seconds to 5 minutes. More preferably, the nin- charge and/or binding affinity. At present, the most com- hydrin reagent and eluted amino acids are allowed to mon technique used to separate amino acids discrimi- react in the heated reaction chamber for a period of from nates on the basis of net charge and is therefore known 30 seconds to 2 minutes, more preferably about 1 minute. as ion exchange chromatography. The present invention 5 It is an advantage of the ninhydrin reagent of the present provides a method which may be used effectively in as- invention that the time required in the reaction chamber sociation with ion exchange chromatography as well as to produce the required colouration is short. other forms of chromatographic separation techniques, [0091] As discussed above, the components of the nin- column and planar, including but not limited to gel filtra- hydrin reagent of this invention react only when subjected tion chromatography, affinity chromatography and high 10 to heating, to produce hydrindantin, which may be con- or low pressure liquid chromatography, involving normal tacted with the amino acids to produce the coloured prod- phase, reverse phase and adsorption separation mech- uct for photometric analysis. In the method of the present anisms. In addition, the present invention provides a invention, the components of the ninhydrin reagent are method which may be used in association with non-chro- heated within the heated colour forming reaction cham- matographic separation techniquessuch as column elec- 15 ber, typically a coil. Hydrindantin is formed in very low trophoresis. Furthermore, the reagent may effectively be concentrations at the very beginning of the colour forming used for techniques not necessarily involving chromato- reaction coil and the hydrindantin concentration increas- graphic separation such as fingerprint visualization and es with time as the reagent continues to be exposed to colour development in tubes or containers. the elevated temperature. In this way, the ninhydrin re- [0086] In one embodiment, the present invention is ap- 20 agent of the present invention may be used on commer- plied as part of an ion exchange chromatography proce- cially available analysers, with no need for modifying their dure, comprising passing the amino acid-containing so- component parts or method of operation. lution through a resin packed ion exchange column to [0092] The components of the ninhydrin reagent may separate the amino acid sample into the respective ami- be introduced into the colour forming reaction chamber no acid components by differences in their migration25 in a number of different ways as described herein below. speeds down the column. The eluted amino acids are [0093] In a first embodiment, ninhydrin, a temperature- then free to react with a colour developing solution ac- dependent reducing agent as described above, and the cording to the method of the present invention. buffer and optionally one or more organic solvents are [0087] As discussed above the colour developing so- pre- mixed to form a homogeneous solution. This homo- lution is comprised of ninhydrin, a temperature-depend- 30 geneous solution is the ninhydrin reagent of the first as- ent reducing agent inactive at a first temperature and pect of the present invention. This is the preferred em- active at a second threshold temperature to reduce nin- bodiment as the mixture may be formed well in advance hydrin to form hydrindantin, an acetate buffer and pref- of its use in the method of the present invention. erably an organic solvent. [0094] More preferably the mixture is drawn from one [0088] The ninhydrin reagent as described above, is 35 or more containers or reservoirs by one or more pumps suitable for use on a standard amino acid analyser. As and delivered to a common zone at any convenient point discussed above, existing amino acid analysers com- during the amino acid analysis procedure. The zone is prise a single heated reaction chamber. This is also situated downstream of the ion exchange column but up- known as the colour forming chamber, as this is where stream of the colour forming reaction chamber. This ar- Ruhemann’s purple is produced after the separation col- 40 rangement is one preferred embodiment, as commercial- umn effluent is mixed with the ninhydrin reagent. ly available analysers typically deliver ninhydrin reagents [0089] The temperatures of the colour forming cham- comprising hydrindantin in this way. Accordingly, the nin- ber and/or concentration of the temperature-dependent hydrin reagent of the present invention may be used on reducing agent are preferably selected in order to avoid standard amino acid analysers with no need for modifying precipitation of the resulting hydrindantin. The tempera- 45 their component parts or their method of operation. ture within the colour forming chamber may be varied [0095] Yet still more preferably, the zone is situated according to the temperature-dependent reducing agent within the colour forming reaction chamber. In this way, employed and the extent to which it is capable of reducing hydrindantin is formed immediately upon heating within ninhydrin at higher temperatures. In general however, the colour forming reaction chamber. the temperature of the colour forming chamber is prefer- 50 [0096] In an alternative embodiment, the components ably in the range of from 50°C to 150°C, more preferably of the ninhydrin reagent are kept separate until shortly from 80°C to 150°C. Still more preferably, the tempera- before heating and contacting with the amino acids. For ture of the colour forming chamber is from 120°C to example, ninhydrin and the temperature-dependent re- 140°C. Yet still more preferably, the temperature of the ducing agent may be drawn from a single or separate colour forming chamber is from 125°C to 135°C. 55 containers or reservoirs by two or more pumps and de- [0090] The ninhydrin reagent and eluted amino acids livered to a common line or mixing zone, where they are are heated within the heated reaction chamber and al- combined to form a mixture. In this embodiment, the buff- lowed to react for a sufficient period of time, for example er and optional one or more organic solvents may each

10 19 EP 2 735 877 B1 20 be drawn from a single or separate containers or reser- cussed, glycine is chosen as the standard ref- voirs to combine with the mixture of ninhydrin and the erence amino acid. compound inactive at room temperature. [0097] Similarly, the common line or mixing zone is typ- 2) In a glass test tube measuring close to 15 cm long ically situated downstream of the ion exchange column 5 and 1.5 cm in outside diameter, add 2.0 ml of solution but upstream of the colour forming reaction chamber. of i) above, 0.20ml of the standard glycine solution This arrangement is preferred as commercially available ii) and 2.8 ml of pure water. analysers typically deliver ninhydrin reagents comprising hydrindantin in this way. Accordingly, the ninhydrin rea- 3) Place the test tube into a liquid heating bath at gent of the present invention may be used on standard 10 100°C and leave undisturbed for 20 minutes. Take amino acid analysers with no need for modifying their out the test tube and cool quickly to room tempera- component parts or their method of operation. ture. [0098] Yet still more preferably, the zone is situated within the colour forming reaction chamber. In this way, 4) Transfer the cooled solution into a 1 cm cuvette hydrindantin is formed immediately upon heating within 15 and measure the absorbance at 570 nm against pure the colour forming reaction chamber. water using a standard spectrophotometer or color- [0099] The amino acid stream may be contacted with imeter. the components or mixture of components at any con- venient point in the procedure. For example, the amino 5) At the same time as the compound is being as- acids may be combined with one of the ninhydrin or tem- 20 sessed for activity, a blank sample should also be perature-dependent reducing agent, prior to forming the performed in case there are amino acid or amine mixture and heating. As an alternative, the amino acids impuritiesin thereagents. Theprocedure is the same may be combined with the mixture of ninhydrin and the as in 2) above except that the glycine standard is temperature-dependent reducing agent prior to heating. omitted and the quantity of pure water is increased As a further alternative, the amino acids may be com- 25 to 3.0 ml. The absorbance is then measured at bined with the components of the reagent after mixing 570nm as per 4) above. and heating has occurred and hydrindantin formed. [0100] As noted above, a range of temperature- de- 6) The actual absorbance produced by the com- pendent reducing agents have been identified using Pro- pound under assessment, hereafter called the cor- tocol 1 and 2, as being inactive at a first temperature and 30 rected absorbance, is obtained by subtracting the active at a second threshold temperature to reduce nin- absorbance of the blank sample from the glycine hydrin to hydrindantin. Protocol 1, Protocol 2 and em- standard absorbance. bodiments of the present invention will now be described in the following examples. [0102] As the degree of activity of compound X is un- 35 known, the method of the above Protocol is preferably EXAMPLES repeated a number of times, each time gradually increas- ing the concentration of compound X. The concentration Example 1: Protocol 1 of compound X is to be increased until the corrected ab- sorbance falls between 0.6 and 0.7. The maximum cor- [0101] Protocol 1 of the present invention is comprised 40 rected absorbance possible when using glycine as a con- of the following steps; trol is between 0.75 and 0.80, so no advantage will nec- essarily be gained in this Protocol assessment by in- 1) Prepare the following solutions: creasing the concentration of the compound any further if a corrected absorbance of 0.6 to 0.7 is achieved. i) A 1 M lithium, sodium or potassium ace-45 [0103] If the corrected absorbance never exceeds tate/acetic acid aqueous buffer at pH 5.2, con- 0.30, this indicates that compound X does not have the taining 2% ninhydrin w/v and a chosen starting degree of activity required for use in the ninhydrin reagent concentration (w/v or v/v) of compound X. It is of the present invention. preferred to start with 0.01% w/v or v/v and grad- [0104] If the corrected absorbance falls below 0.30 but ually increase the concentration in stages. High 50 increasing the concentration of compound X causes it to concentrations of water soluble solids or liquids, precipitate out of solution, or causes some other adverse particularly approaching 50% and above may effect, this indicates that compound X does not have the be difficult to evaluate, due to solubility problems properties required for use in the ninhydrin reagent of the or other adverse effects after preparation or present invention. when used on an analytical system. 55 Example 2: Protocol 2 ii) A standard solution of glycine at a concentra- tion of 1.0 mM in pure water. As previously dis- [0105] Provided, compound X is sufficiently active at

11 21 EP 2 735 877 B1 22 elevated temperatures to meet the requirements of Pro- using a flow cell with 1 cm path length. tocol 1 set out in Example 1 above, the following test may be used to assess the long term stability of a ninhydrin 4) On day one calculate the corrected absorbance reagent comprising compound X at room temperature produced by compound X. This is obtained by sub- and in the presence of air. 5 tracting the absorbance of the blank sample from the [0106] A ninhydrin reagent is prepared in a similar way glycinesample absorbance,as described in Protocol to Protocol 1 of Example 1 and stored at room tempera- 1 of Example 1 ture in the presence of air. At set time intervals, a sample is taken from the ninhydrin reagent, mixed with an amino 5) On day one, after steps 1 to 4 have been com- acid solution and heated to 100°C for a set time. The10 pleted, store the reagent in a stoppered bottle at 20 amount of Ruhemann’s purple produced, if any, is meas- °C in the dark and exposed to the air. This is achieved ured in terms of an absorbance value. The rate of de- by leaving a large air gap approximately the same crease in intensity of coloration (absorbance) in succes- volume as the reagent in the bottle. sive samples indicates whether the long term stability of compound X is adequate for forming part of the ninhydrin 15 6) Every two to four weeks, take a sample of the reagent of the present invention. reagent and repeat steps 2 and 3. Each time, calcu- [0107] As per Protocol 1, glycine is used as the stand- late the corrected absorbance at 570nm as per step ard amino acid as the relative sensitivities of other amino 4 and compare it with that taken at day one. acids and amine compounds will be similar whatever re- agent is used. 20 Example 3 -Chromatography Examples [0108] Protocol 2 of the present invention is comprised of the following steps; [0109] The relative sensitivities of a number of ninhy- drin reagents comprising different temperature-depend- 1) On day One prepare the following solutions: ent reducing agents has been assessed using a standard 25 amino acid analyser. As per the test Protocol and batch i) A standard 1.0 mM solution of glycine in water. stability test, glycine was used as the reference amino acid. ii) A ninhydrin reagent comprising a 1 M lithium, [0110] As discussed above, the ninhydrin reagent of sodium or potassium acetate/acetic acid aque- the present invention may optionally comprise an organic ous buffer at pH 5.2, containing 2% ninhydrin 30 solvent or mixture of organic solvents. Accordingly, for w/v and a chosen concentration (w/v or v/v) of the sake of comparison, the relative sensitivities of these compound X. The concentration of compound ninhydrin reagents have been assessed both in the pres- X is preferably similar to that identified in Proto- ence and in the absence of an organic solvent. As noted col 1 as the concentration at which a corrected above, if an organic solvent is used, the total concentra- absorbance of between 0.4 and 0.7 is achieved. 35 tion will preferably vary from 35% to 55% according to 250 ml of reagent should be enough volume to the nature of the organic solvent used. In these experi- last the full length of the stability trial. ments, the temperature-dependent reducing agents were all assessed in the presence of 40 % Ethylene Gly- 2) On day one, to a standard test tube, add in suc- col. cession, 2.0 ml of the ninhydrin reagent (ii), 0.20 ml 40 [0111] Chromatography experiments were conducted of the glycine standard (i) and 2.8 ml of pure water. several times in relation to each of the temperature-de- Mix and place the test tube in a liquid heating bath pendent reducing agents, until their sensitivity was opti- at 100C° and leave undisturbed for 35 minutes. Take mised. This was achieved by varying the concentration out the test tube and cool quickly to room tempera- of the temperature-dependent reducing agents and/or ture. Measure the absorbance of the test tube solu- 45 the reaction temperature. tion in a spectrophotometer at 570 nm against pure [0112] The starting concentration was that which was water using a flow cell with 1 cm path length. found to satisfy the conditions of the test Protocol as de- scribed above. As noted above, the starting temperature 3) On day one, prepare a blank test tube sample within the colour forming chamber may be varied accord- comprising 2.0 ml of reagent (ii) and 3.0 ml of pure 50 ing to the temperature-dependent reducing agent em- water. This will take into account any amino acid or ployed but is preferably in the region of from 125°C to amine type impurities that could be present in the 135°C. components used to make up the reagents. Mix and [0113] For the chromatography examples shown here, place the blank test tube in a heating bath at 100C° the relative sensitivities for the different reagent compo- and leave undisturbed for 20 minutes. Take out the 55 sitions are compared in terms of peaks heights for glycine test tube and cool quickly to room temperature. in units of mAU. Thus, the chromatographic conditions Measure the absorbance of the test tube solution in of retention time and peak widths (that is the peak width a spectrophotometer at 570 nm against pure water at half height) are constant for all the analyses (See chro-

12 23 EP 2 735 877 B1 24 matographic conditions below). [0122] In the presence of 40% ethylene glycol the nin- [0114] The following instrument conditions were used: hydrin reagent comprising 2% glucose w/v provided a peak height of 97 mAU, at a reaction temperature of 131 Chromatographic mode - Isocratic °C. 5 [0123] In the absence of ethylene glycol, the ninhydrin Column flow rate - 0.45 ml/min reagent comprising 2% glucose w/v provided a peak height of 79 mAU at a reaction temperature of 133 °C. Ninhydrin reagent flow rate - 0.30 ml/min [0124] The above results show a large increase in ac- tivity from glucose to sodium formate to fructose as Temperature of heated reaction chamber - Variable 10 shown by the percentage concentration required for glu- between 125 and 135°C cose and fructose to achieve similar sensitivities which varies from 2% to 0.2% w/v respectively. The percentage Reaction time - 60 seconds concentration required for achieving the desired sensi- tivity is therefore expected to vary depending upon the Injection volume - 20 ml 15 compound(s) used to form the temperature-dependent reducing agent. Optimisation of factors such as the per- Amount of glycine injected - 2 nmoles centage concentration of the temperature-dependent re- ducing agent, percentage concentration of organic sol- Retention time - 5.0 minutes vent if any, and the temperature of the reaction chamber, 20 during use on standard amino acid analysers, will identify Peak width at half height - 0.26 minutes the optimal percentage concentration for all compounds passing Protocol 1 and 2. Sensitivity scale - mAU (1 mAU is equivalent to 0.001 [0125] Further, the results show that in the absence of absorbance) an organic solvent the sensitivities of the temperature- 25 dependent reducing agents decreases. However, it can Example 3.1 - Fructose beseen that thisdecrease in activity doesnot significantly affect the sensitivity of the analytical method. [0115] The sensitivity of a ninhydrin reagent compris- ing 0.20% w/v fructose as the temperature-dependent reducing agent was assessed using a standard amino 30 Claims acid analyser under the conditions specified above. [0116] In the presence of 40 % ethylene glycol, the 1. A ninhydrin reagent for use in the chromatographic peak height was identified as 112mAU at a reaction tem- analysis of amino acids, the ninhydrin reagent com- perature of 131 °C. prising: [0117] In the absence of ethylene glycol, the ninhydrin 35 reagent comprising 0.20% w/v fructose provided a peak ninhydrin; height of 88 mAU at a reaction temperature of 132 °C an aqueous buffer, wherein the buffer maintains the pH at a value between 4.5 and 5.5; and Example 3.2 - Sodium formate a temperature-dependent reducing agent, 40 which agent is inactive in the reduction of nin- [0118] The sensitivity of a ninhydrin reagent compris- hydrin at a first temperature, wherein the first ing 1% sodium formate w/v as the temperature-depend- temperature isup to30°C, and active inreducing entreducing agent was assessedusing a standard amino ninhydrin to hydrindantin at a second tempera- acid analyser under the conditions specified above. ture, wherein the second temperature is higher [0119] In the presence of 40 % ethylene glycol, the 45 than the first temperature. ninhydrin reagent comprising 1% sodium formate provid- ed a peak height of 126 mAU at a reaction temperature 2. The ninhydrin reagent according to claim 1, wherein of 133 °C. the reagent is substantially free from hydrindantin [0120] In the absence of ethylene glycol, the ninhydrin reagent comprising 1% sodium formate provided a peak 50 3. The ninhydrin reagent according to either of claims height of 107 mAU at a reaction temperature of 133 °C. 1 or 2, wherein the second temperature is at least 100°C Example 3.3 - Glucose 4. The ninhydrin reagent according to any preceding [0121] The sensitivity of a ninhydrin reagent compris- 55 claim, wherein the temperature-dependent reducing ing 2% glucose w/v as the temperature-dependent re- agent is a compound meeting the standards of re- ducing agent was assessed using a standard amino acid ducing activity at the second temperature of Protocol analyser under the conditions specified above. 1 of Example 1, more preferably wherein the tem-

13 25 EP 2 735 877 B1 26

perature-dependent reducing agent is a compound formic acid or a salt thereof; or wherein the temper- meeting the standards of reducing activity at the sec- ature-dependent reducing agent comprises one or ond temperature according to Protocol 1, with which more carboxylic acids comprising at least one addi- a minimum absorbance of 0.4 is observed. tional reducing group or a salt thereof; wherein if salts 5 are employed they are preferably a Group I or Group 5. The ninhydrin reagent according to any preceding II metal salt of the carboxylic acid. claim, wherein the first temperature is from 10°C to 20°C. 9. The ninhydrin reagent according to any preceding claim, wherein the temperature-dependent reducing 6. The ninhydrin reagent according to any preceding 10 agent comprises one or more compounds having claim, wherein the temperature-dependent reducing one or more aldehyde and/or ketone groups, more agent is a compound meeting the standards of re- preferably, wherein the temperature-dependent re- ducing activity at the first temperature according to ducing agent comprises acetone. Protocol 2 of Example 2, preferably wherein the tem- perature-dependent reducing agent is a compound 15 10. Theninhydrin reagent accordingto claim 28, wherein having an activity of at least 50% of its initial activity the temperature-dependent reducing agent com- after 3 months, more preferably wherein the temper- prises an inorganic compound of sulphur in a low ature-dependent reducing agent is a compound hav- oxidation state or a phosphorus oxyacid in a low ox- ing an activity of at least 50% of its initial activity after idation state, more preferably wherein the tempera- 6 months, more preferably still wherein the temper- 20 ture-dependent reducing agent comprises a sulfite, ature-dependent reducing agent is a compound hav- thiosulfate, a phosphite and/or a hypophosphite. ing an activity of at least 50% of its initial activity after 12 months, more preferably still wherein the temper- 11. The ninhydrin reagent according to any preceding ature-dependent reducing agent is a compound hav- claim, wherein the concentration of the temperature- ing an activity of at least 50% of its initial activity after 25 dependent reducing agent is from 0.01% to 75% w/v 24 months. or v/v, more preferably wherein the concentration of the temperature-dependent reducing agent is from 7. The ninhydrin reagent according to any preceding 0.01% to 20% w/v or v/v. claim, wherein the temperature-dependent reducing agent comprises one or more saccharides, prefera- 30 12. Theninhydrin reagent accordingto claim 32, wherein bly wherein the temperature-dependent reducing the concentration of ninhydrin in the ninhydrin rea- agent comprises one or more monosaccharide’s se- gent is from 0.5 to 3% w/v or v/v, more preferably, lected from: Glucose, Fructose, Glyceraldehyde, wherein the concentration of ninhydrin in the ninhy- Galactose, Ribose, Xylose, Erythrose, Threose, drin reagent is from 1 to 2.5% w/v or v/v, more pref- Lyxose, Arabinose, Allose, Altrose, Mannose, Gu- 35 erably still wherein the concentration of ninhydrin in lose, Idose, Talose and L-Glycero-D-manno-hep- the ninhydrin reagent is about 2% w/v or v/v. tose, Dihydroxyacetone, Erythrulose, Ribulose, Xy- lulose, Psicose, Sorbose, Tagatose, Sedohepta- 13. The ninhydrin reagent according to any preceding lose, or a mixture thereof; and/or wherein the tem- claim, wherein the buffer is an aqueous buffer, pref- perature-dependent reducing agent comprises one 40 erably wherein the buffer comprises a weak acid and or more disaccharides selected from: Lactose, Mal- one of its conjugate bases/salts, more preferably tose, Trihalose, Cellobiose, Kojibiose, Nigerose, Iso- wherein the buffer comprises an organic acid, more maltose, Sophorose, Laminaribiose, Gentiobiose, preferably still wherein the buffer comprises acetic Turanose, Maltulose, Palatinose, Gentiobiulose, acid, ethanoic acid or propanoic acid. Mannobiose, Melibiose, Melibiulose, Rutinose, Xy- 45 lobiose or a mixture thereof; and/or wherein the tem- 14. The ninhydrin reagent according to claim, further perature-dependent reducing agent comprises one comprising one or more organic solvents, preferably or more longer chain polysaccharides selected from wherein the organic solvent is selected from dimeth- Stachyose, Dextrin, Maltodextrin, or a mixture there- ylsulfoxide, ethylene glycol, propylene glycol, sul- of. 50 folane, carbitol, propylene glycol monomethyl ether, methylcellosolve and methanol. 8. The ninhydrin reagent according to any preceding claim, wherein the temperature-dependent reducing 15. Theninhydrin reagent accordingto claim 14, wherein agentcomprises one or more carboxylic acids and/or the concentration of organic solvent is from 10% to a salt thereof, preferably wherein the temperature- 55 75% v/v, preferably wherein the concentration of or- dependent reducing agent comprises one or more ganic solvent is from 25% to 65% v/v, more prefer- monofunctional carboxylic acids or a salt thereof, ably wherein the concentration of organic solvent is more preferably still wherein the carboxylic acid is from 35% to 55% v/v.

14 27 EP 2 735 877 B1 28

16. A chromatographic method of analysing an amino Standards von Reduktionsaktivität bei der ersten acid, the method comprising: Temperatur gemäß Protokoll 2 von Beispiel 2 erfüllt, wobei das temperaturabhängige Reduktionsmittel i) providing a ninhydrin reagent according to any vorzugsweise eine Verbindung ist, die nach 3 Mo- of claims 1 to 15; 5 naten eine Aktivität von mindestens 50 % ihrer an- ii) heating the ninhydrin reagent to at least the fänglichen Aktivität aufweist, wobei das temperatur- second temperature to form a hydrindantin-con- abhängige Reduktionsmittel insbesondere eine Ver- taining mixture; and bindung ist, die nach 6 Monaten eine Aktivität von iii) contacting the hydrindantin-containing mix- mindestens 50 % ihrer anfänglichen Aktivität auf- ture with the nitrogen-containing compound; 10 weist, wobei das temperaturabhängige Reduktions- mittel vornehmlich eine Verbindung ist, die nach 12 preferably wherein the method further comprises an- Monaten eine Aktivität von mindestens 50 % ihrer alysing the colour of the product of step (iii). anfänglichen Aktivität aufweist, wobei das tempera- turabhängige Reduktionsmittel wünschenswerter- 15 weise eine Verbindung ist, die nach 24 Monaten eine Patentansprüche Aktivität von mindestens 50 % ihrer anfänglichen Ak- tivität aufweist. 1. Ninhydrin-Reagens zur Verwendung bei der chro- matografischen Analyse von Aminosäuren, wobei 7. Ninhydrin-Reagens nach einem der vorhergehen- das Ninhydrin-Reagens umfasst: 20 den Ansprüche, wobei das temperaturabhängige Reduktionsmittel ein oder mehrere Saccharide um- Ninhydrin; fasst, wobei das temperaturabhängige Reduktions- einen wässrigen Puffer, wobei der Puffer den mittel vorzugsweise ein oder mehrere Monosaccha- pH auf einem Wert von 4,5 bis 5,5 hält; und ride umfasst, die ausgewählt sind aus: Glucose, ein temperaturabhängiges Reduktionsmittel,25 Fructose, Glyceraldehyd, Galactose, Ribose, Xylo- welches Mittel bei der Reduktion von Ninhydrin se, Erythrose, Threose, Lyxose, Arabinose, Allose, bei einer ersten Temperatur inaktiv ist, wobei Altrose, Mannose, Gulose, Idose, Talose und L-Gly- die erste Temperatur bis zu 30 °C ist, und beim cero-D-manno-heptose, Dihydroxyaceton, Erythru- Reduzieren von Ninhydrin zu Hydrindantin bei lose, Ribulose, Xylulose, Psicose, Sorbose, Tagato- einer zweiten Temperatur aktiv ist, wobei die 30 se, Sedoheptalose oder einer Mischung davon; zweite Temperatur höher als die erste Tempe- und/oder wobei das temperaturabhängige Redukti- ratur ist. onsmittel ein oder mehrere Disaccharide umfasst, die ausgewählt sind aus: Lactose, Maltose, Trihalo- 2. Ninhydrin-Reagens nach Anspruch 1, wobei das Re- se, Cellobiose, Kojibiose, Nigerose, Isomaltose, So- agens im Wesentlichen frei von Hydrindantin ist. 35 phorose, Laminaribiose, Gentiobiose, Turanose, Maltulose, Palatinose, Gentiobiulose, Mannobiose, 3. Ninhydrin-Reagens nach einem der Ansprüche 1 Melibiose, Melibiulose, Rutinose, Xylobiose oder ei- oder2, wobei diezweite Temperatur mindestens 100 ner Mischung davon; und/oder wobei das tempera- °C beträgt. turabhängige Reduktionsmittel ein oder mehrere 40 langkettige Polysaccharide umfasst, die aus Stachy- 4. Ninhydrin-Reagens nach einem der vorhergehen- ose, Dextrin, Maltodextrin oder einer Mischung da- den Ansprüche, wobei das temperaturabhängige von ausgewählt sind. Reduktionsmittel eine Verbindung ist, welche die Standards von Reduktionsaktivität bei der zweiten 8. Ninhydrin-Reagens nach einem der vorhergehen- Temperatur von Protokoll 1 von Beispiel 1 erfüllt, wo- 45 den Ansprüche, wobei das temperaturabhängige bei das temperaturabhängige Reduktionsmittel vor- Reduktionsmittel eine oder mehrere Carbonsäuren zugsweise eine Verbindung ist, welche die Stan- und/oder ein Salz davon umfasst, wobei das tempe- dards von Reduktionsaktivität bei der zweiten Tem- raturabhängige Reduktionsmittel vorzugsweise eine peratur gemäß Protokoll 1 erfüllt, bei der eine Min- oder mehrere monofunktionelle Carbonsäuren oder destabsorbanz von 0,4 zu beobachten ist. 50 ein Salz davon umfasst, wobei die Carbonsäure ins- besondere Ameisensäure oder ein Salz davon ist; 5. Ninhydrin-Reagens nach einem der vorhergehen- oder wobei das temperaturabhängige Reduktions- den Ansprüche, wobei die erste Temperatur 10 °C mittel eine oder mehrere Carbonsäuren umfasst, die bis 20 °C beträgt. mindestens eine zusätzliche Reduktionsgruppe 55 oder ein Salz davon umfassen; wobei, wenn Salze 6. Ninhydrin-Reagens nach einem der vorhergehen- eingesetzt werden, es sich dabei vorzugsweise um den Ansprüche, wobei das temperaturabhängige ein Gruppe I oder Gruppe II Metallsalz der Carbon- Reduktionsmittel eine Verbindung ist, welche die säure handelt.

15 29 EP 2 735 877 B1 30

9. Ninhydrin-Reagens nach einem der vorhergehen- einem der Ansprüche 1 bis 15; den Ansprüche, wobei das temperaturabhängige ii) Erwärmen des Ninhydrin-Reagens auf min- Reduktionsmittel eine oder mehrere Verbindungen destens die zweite Temperatur, um ein Hydrind- mit einer oder mehreren Aldehyd- und/oder Keton- antin-haltiges Gemisch zu bilden; und gruppen umfasst, wobei das temperaturabhängige 5 iii) In-Kontakt-Bringen des Hydrindantin-halti- Reduktionsmittel vorzugsweise Aceton umfasst. gen Gemisches mit einer stickstoffhaltigen Ver- bindung; 10. Ninhydrin-Reagens nach Anspruch 28, wobei das temperaturabhängige Reduktionsmittel eine anor- wobei das Verfahren vorzugsweise ferner ein Ana- ganische Verbindung von Schwefel in einem Zu- 10 lysieren der Farbe des Produkts von Schritt (iii) um- stand geringer Oxidation oder eine Phosphor-Oxo- fasst. säure in einem Zustand geringer Oxidation umfasst, wobei das temperaturabhängige Reduktionsmittel vorzugsweise ein Sulfit, ein Thiosulfat, ein Phosphit Revendications und/oder ein Hypophosphit umfasst. 15 1. Réactif à la ninhydrine destiné à être utilisé dans 11. Ninhydrin-Reagens nach einem der vorhergehen- l’analyse chromatographique des acides aminés, le den Ansprüche, wobei die Konzentration des tem- réactif à la ninhydrine comprenant : peraturabhängigen Reduktionsmittels 0,01 % bis 75 % w/v oder v/v beträgt, wobei die Konzentration des 20 de la ninhydrine ; temperaturabhängigen Reduktionsmittels vorzugs- un tampon aqueux dans lequel le tampon main- weise 0,01 % bis 20 % w/v oder v/v beträgt. tient le pH à une valeur comprise entre 4,5 et 5,5 ; et 12. Ninhydrin-Reagens nach Anspruch 32, wobei die un agent réducteur sensible à la température, Konzentration von Ninhydrin im Ninhydrin-Reagens 25 ledit agent étant inefficace dans la réduction de 0,5 bis 3 % w/v oder v/v beträgt, wobei die Konzen- la ninhydrine à une première température, la tration von Ninhydrin im Ninhydrin-Reagens vor- première température allant jusqu’à 30 °C, et zugsweise 1 bis 2,5 % w/v oder v/v beträgt, wobei efficace dans la réduction de la ninhydrine pour die Konzentration von Ninhydrin im Ninhydrin-Rea- donner de l’hydrindantin à une seconde tempé- gens insbesondere etwa 2 % w/v oder v/v beträgt. 30 rature, la seconde température étant supérieure à la première température. 13. Ninhydrin-Reagens nach einem der vorhergehen- den Ansprüche, wobei der Puffer ein wässriger Puf- 2. Réactif à la ninhydrine selon la revendication 1, dans ferist, wobei der Puffer vorzugsweiseeine schwache lequel le réactif est essentiellement exempt d’hydrin- Säure und eine/s ihrer konjugierten Basen/Salze35 dantin. umfasst, wobei der Puffer insbesondere eine orga- nische Säure umfasst, wobei der Puffer vornehmlich 3. Réactif à la ninhydrine selon l’une quelconque des Essigsäure, Ethansäure oder Propansäure umfasst. revendications 1 et 2, dans lequel la seconde tem- pérature est d’au moins 100 °C. 14. Ninhydrin-Reagens nach Anspruch, ferner umfas- 40 send ein oder mehrere organische Lösungsmittel, 4. Réactif à la ninhydrine selon l’une quelconque des wobei das organische Lösungsmittel vorzugsweise revendications qui précèdent, dans lequel l’agent ré- aus Dimethylsulfoxid, Ethylenglycol, Propylenglycol, ducteur sensible à la température est un composé Sulfolan, Carbitol, Propylenglycolmonomethylether, qui répond aux normes de l’activité réductrice à la Methyl-Cellosolve und Methanol ausgewählt ist. 45 seconde température du protocole 1 de l’exemple 1, de préférence dans lequel l’agent réducteur sensible 15. Ninhydrin-Reagens nach Anspruch 14, wobei die à la température est un composé qui répond aux Konzentration von organischem Lösungsmittel 10 normes de l’activité réductrice à la seconde tempé- bis 75 % v/v beträgt, wobei die Konzentration von rature selon le protocole 1, avec lequel on observe Ninhydrin von organischem Lösungsmittel vorzugs- 50 une absorbance minimale de 0,4. weise 25 bis 65 % v/v beträgt, wobei die Konzentra- tion von organischem Lösungsmittel insbesondere 5. Réactif à la ninhydrine selon l’une quelconque des etwa 35 % bis 55 % v/v beträgt. revendications qui précèdent, dans lequel la premiè- re température est comprise entre 10 °C et 20 °C. 16. Chromatografisches Verfahren zur Analyse einer 55 Aminosäure, wobei das Verfahren umfasst: 6. Réactif à la ninhydrine selon l’une quelconque des revendications qui précèdent, dans lequel l’agent ré- i) Bereitstellen eines Ninhydrin-Reagens nach ducteur sensible à la température est un composé

16 31 EP 2 735 877 B1 32

qui répond aux normes de l’activité réductrice à la ce, un sel métallique du groupe I ou du groupe II de première température selon le protocole 2 de l’exem- l’acide carboxylique. ple 2, de préférence dans lequel l’agent réducteur sensible à la température est un composé ayant une 9. Réactif à la ninhydrine selon l’une quelconque des activité au moins égale à 50 % de son activité initiale 5 revendications qui précèdent, dans lequel l’agent ré- après 3 mois, mieux encore dans lequel l’agent ré- ducteur sensible à la température comprend un ou ducteur sensible à la température est un composé plusieurs composés comprenant un ou plusieurs ayant une activité au moins égale à 50 % de son groupes aldéhyde et/ou cétone, de préférence dans activité initiale après 6 mois, de manière particuliè- lequel l’agent réducteur sensible à la température rement préférée dans lequel l’agent réducteur sen- 10 comprend de l’acétone. sible à la température est un composé ayant une activité au moins égale à 50 % de son activité initiale 10. Réactif à la ninhydrine selon la revendication 28, après 12 mois, de manière plus avantageuse encore dans lequel l’agent réducteur sensible à la tempéra- dans lequel l’agent réducteur sensible à la tempéra- ture comprend un composé inorganique du soufre à ture est un composé ayant une activité au moins 15 un faible degré d’oxydation ou un oxyacide phos- égale à 50 % de son activité initiale après 24 mois. phoreux à un faible degré d’oxydation, de préférence dans lequel l’agent réducteur sensible à la tempéra- 7. Réactif à la ninhydrine selon l’une quelconque des ture comprend un sulphite, un thiosulfate, un phos- revendications qui précèdent, dans lequel l’agent ré- phite et/ou un hypophosphite. ducteur sensible à la température comprend un ou 20 plusieurs saccharides, de préférence dans lequel 11. Réactif à la ninhydrine selon l’une quelconque des l’agent réducteur sensible à la température com- revendicationsqui précèdent,dans lequella concen- prend un ou plusieurs monosaccharides sélection- tration de l’agent réducteur sensible à la température nés parmi les éléments suivants : glucose, fructose, est comprise entre 0,01 % et 75 % p/v ou v/v, de glycéraldéhyde, galactose, ribose, xylose, érythro- 25 préférence dans lequel la concentration de l’agent se, thréose, lyxose, arabinose, allose, altrose, man- réducteur sensible à la température est comprise en- nose, gulose, idose, talose et L-glycéro-D-manno- tre 0,01 % et 20 % p/v ou v/v. heptose, dihydroxyacétone, érythrulose, ribulose, xylulose, psicose, sorbose, tagatose, sédoheptalo- 12. Réactif à la ninhydrine selon la revendication 32, se,ou un mélange de cesderniers ; et/oudans lequel 30 dans lequel la concentration de la ninhydrine dans l’agent réducteur sensible à la température com- le réactif à la ninhydrine est comprise entre 0,5 et 3 prend un ou plusieurs disaccharides sélectionnés % p/v ou v/v, de préférence dans lequel la concen- parmi les éléments suivants : lactose, maltose, tri- tration de la ninhydrine dans le réactif à la ninhydrine halose, cellobiose, kojibiose, nigérose, isomaltose, est comprise entre 1 et 2,5 % p/v ou v/v, mieux en- sophorose, laminaribiose, gentiobiose, turanose, 35 core dans lequel la concentration de la ninhydrine maltulose, palatinose, gentiobiulose, mannobiose, dans le réactif à la ninhydrine est d’environ 2 % p/v mélibiose, mélibiulose, rutinose, xylobiose ou un ou v/v. mélange de ces derniers ; et/ou dans lequel l’agent réducteur sensible à la température comprend un ou 13. Réactif à la ninhydrine selon l’une quelconque des plusieurs polysaccharides à chaîne plus longue sé- 40 revendications qui précèdent, dans lequel le tampon lectionnés parmi les éléments suivants : stachyose, est un tampon aqueux, de préférence dans lequel le dextrine, maltodextrine ou un mélange de ces der- tampon comprend un acide faible ou l’une de ses niers. bases conjuguées/l’un de ses sels, mieux encore dans lequel le tampon comprend un acide organi- 8. Réactif à la ninhydrine selon l’une quelconque des 45 que, de manière particulièrement préférée dans le- revendications qui précèdent, dans lequel l’agent ré- quel le tampon comprend de l’acide acétique, de ducteur sensible à la température comprend un ou l’acide éthanoïque ou de l’acide propanoïque. plusieurs acides carboxyliques et/ou un sel de ces derniers, de préférence dans lequel l’agent réduc- 14. Réactif à la ninhydrine selon la revendication, com- teur sensible à la température comprend un ou plu- 50 prenant en outre un ou plusieurs solvants organi- sieurs acides carboxyliques monofonctionnels ou un ques,de préférence dans lequel le solvant organique sel de ces derniers, mieux encore dans lequel l’acide est sélectionné parmi les éléments suivants : dimé- carboxylique est l’acide formique ou un sel de ce thylsulfoxyde, éthylène glycol, propylène glycol, sul- dernier ; ou dans lequel l’agent réducteur sensible à folane, carbitol, monométhyléther de propylène gly- la température comprend un ou plusieurs acides car- 55 col, méthyl cellosolve et méthanol. boxyliques comprenant au moins un groupe réduc- teur supplémentaire ou un sel de ce dernier ; dans 15. Réactif à la ninhydrine selon la revendication 14, lequel, si des sels sont utilisés, ils sont, de préféren- dans lequel la concentration du solvant organique

17 33 EP 2 735 877 B1 34

est comprise entre 10 % et 75 % v/v, de préférence dans lequel la concentration du solvant organique est comprise entre 25 % et 65 % v/v, mieux encore dans lequel la concentration du solvant organique est comprise entre 35 % et 55 % v/v. 5

16. Procédé chromatographique d’analyse d’un acide aminé, le procédé comprenant :

i) la fourniture d’un réactif à la ninhydrine selon 10 l’une quelconque des revendications 1 à 15 ; ii) le chauffage du réactif à la ninhydrine jusqu’à au moins la seconde température pour former un mélange contenant de l’hydrindantin ; et iii) la mise en contact du mélange contenant de 15 l’hydrindantin avec le composé contenant de l’azote ;

de préférence dans lequel le procédé comprend en outre l’analyse de la couleur du produit de l’étape (iii). 20

25

30

35

40

45

50

55

18 EP 2 735 877 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 3632496 A [0011] • US 4274833 A [0018] • US 4359323 A [0012] • GB 1349941 A [0024] • US 3778230 A [0015] [0016] [0017] [0018] • CN 102618621 A [0025]

Non-patent literature cited in the description

• SPACKMAN DH ; STEIN WH ; MOORE S. Automat- ic recording apparatus for use in the chromatography of amino acids. Anal Chem, 1958, vol. 30, 1190-1206 [0003]

19