Novel hantzsch reagents and uses thereof

Abstract

The present invention generally relates to the field of metabolite analysis as it applies to compounds, formulations and methods which can be used to detect metabolites such as formaldehyde and methanol. More particularly, the present invention pertains to β-keto amide compounds which are particularly useful as superior and sufficiently chemoselective detection reagent in the detection of metabolites, notable formaldehyde and related compounds, such as methanol. The present invention also pertains to methods for detecting formaldehyde or methanol, as well as to formulations and kits comprising the β-keto amide compounds, and to the use thereof in the detecting formaldehyde or methanol.

Description

[0001] August 29, 2025

[0002] Roche Diagnostics GmbH R74633PC

[0003] Novel Hantzsch reagents and uses thereof

[0004] Field of the invention

[0005] The present invention generally relates to the field of metabolite analysis as it applies to compounds, formulations and methods which can be used to detect metabolites such as formaldehyde and methanol. More particularly, the present invention pertains to p-keto amide compounds which are particularly useful as superior and sufficiently chemoselective detection reagent in the detection of metabolites, notable formaldehyde and related compounds, such as methanol. The present invention also pertains to methods for detecting formaldehyde or methanol, as well as to formulations and kits comprising the p-keto amide compounds, and to the use thereof in the detecting formaldehyde or methanol.

[0006] Background of the invention

[0007] Formaldehyde (also known as methanal) is on the one hand an important bulk chemical in processing, such as in manufacturing of a variety of chemicals like drugs, biocides, disinfectants, plastic products, building materials; and on the other hand a harmful contaminant produced in the degradation of organic chemicals and materials, food components or generated in combustion, roasting and curing (with smoke) processes.

[0008] Although, formaldehyde is endogenously produced in metabolism of living organisms, formaldehyde exhibits multiple toxic mode of actions in most living beings, is carcinogenic and harmful for the environment in the near-term due to its promiscous reactivity. There is big interest in its sensitive and reliable detection and control. Formaldehyde detection can also be used as an indicator reaction if it is carried out after an initial enzymatic or chemical reaction (oxidation, hydrolysis, ...) that releases formaldehyde. Such a method can be relevant, for example, in food analysis (distilleries, fruit / vegetable products, ...), in carbohydrate detection or in the analysis of biofuels or in fermentation processes.

[0009] Spectrophotometric or spectrofluorometric detection in combination with chemoselective derivatization methods generally hold good potential for fast, sensitive and automatable analyte detection. There are several reagents and methodologies that have been developed so far for formaldehyde detection, utilizing a Hantzsch-like-reactions (such as Sawicki reagent, Nash reagent, and Flural-P), condensation-type reactions (with Chromotropic acid, Hydrazine) or enzymatic detection (formaldehyde dehydrogenase). The current detection reagents have, however, some shortcomings in that they prevent and impede the use for automatization and in high throughput settings, such as when using the Cedex® analyzer family or other spectrophotometers I spectrofluorometers: current formaldehyde detection reagents (Nash reagent, Sawicki reagent, Fluoral-P) or enzymes generally possess a low shelf life or special storage conditions due to their tendency to decomposition, as well as sensitivity toward air, light, water and / or heat; a different approach for formaldehyde detection reagents to circumvent the former aspect is the preparation by mixing I dissolving one or more compounds in a solvent in advance to the detection reaction; however, these reagents fail to reach shelf life that satisfy the use for a prolonged time (1-2 weeks) within the aqueous or organic medium; reagents can be volatile, flammable, have the tendency to form explosive vapor / air mixtures (acetylacetone); reagents might possess concerning toxicities (acetylacetone, hydrazine derivatives) requiring special protective measures for the user such as proper ventilation, protective equipment and special storage and waste management; non-polar reagents ( / V-Acetoacetanilide) require a high content of organics co-solvent in the detection reaction, making the compatibility and measurement in aqueous systems challenging;

[0010] - they are reagents that might require a pH special (acidic or alkaline), heat incubation, the use of additional corrosive reagents (i.e. chromotropic acid) or special equipment, all of which making further preparation steps necessary.

[0011] Other reagents, such as dinitrophenylhydrazine or MTBH, are non-specific for formaldehyde and react to a broader scope of aldehydes I ketones or other electrophilic functional groups.

[0012] Accordingly, it is an objective of the present invention to provide new and advantageous reagents for the detection of formaldehyde and related compounds.

[0013] Summary of the invention

[0014] The above objective has been solved by the present inventors, who have identified p-keto amide compounds as superior and sufficiently chemoselective detection reagent in the detection of formaldehyde and related compounds, such as methanol. These p-keto amide compounds are collectively referred to herein as compound(s) of general formula (I), of which N-(acetoacetyl)anthranilic acid is a particularly preferred example. A compound of general formula (I) when reacted with formaldehyde in the presence of a nitrogen donor, such as ammonia or an ammonium salt, results in the production of a dihydropyridine. This type of reaction is generally known as Hantzsch pyridine synthesis or Hantzsch reaction. The resulting dihydropyridine, which is collectively referred to herein as compound(s) of general formula (II), can then be detected, e.g. spectrophotometrically, and thus may server as a suitable dye in the detection of formaldehyde and related compounds, such as methanol.

[0015] The compound(s) of general formula (I) when used as a Hantzsch reagent has shown to be superior over other p-ketocarbonyl compounds because of its unusually high solubility in aqueous solutions (higher than 10 % w / w), superior kinetics and a high signal in the corresponding reaction with formaldehyde. In addition, the compound(s) of general formula (I) displayed a good stability when compared with other representatives of the classes.

[0016] The present inventors furthermore identified optimized formulations of the Hantzsch reagent made of the compound(s) of general formula (I) regarding signal, kinetics and shelf life.

[0017] The present invention can thus be summarized by the following items.

[0018] 1. Use of a compound of general formula (I) or a salt thereof in a Hantzsch reaction wherein

[0019] R1is -H or -Me; at least one of R2, R3, R4, R5and R6is a carboxylic acid substituent ( COOH), preferably is one carboxylic acid in the R2or R6position; and any one of R2, R3, R4, R5and R6, if not equalling to COOH, is each independently selected from the group consisting of - H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7(CH2)i. 6COOH, -CONR7(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; and

[0020] R7is, if applicable, -H or -Me, preferably is -H.

[0021] 2. Use according to item 1 , wherein the compound of general formula (I) is a compound of formula (la) wherein

[0022] R1is -H or -Me;

[0023] R2, R3, R4, and R5are each independently selected from the group consisting of -H, - OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEts+, -CONH2, -CONR7(CH2)I.6COOH, -CONR7(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; and

[0024] R7is, if applicable, -H or -Me, preferably is -H. Use according to item 1 , wherein the compound of general formula (I) is a compound of formula (lb) wherein R1is -H or -Me. Use according to item 1 , wherein the compound of general formula (I) is a compound of formula (Ic) wherein R1is -H or -Me.

[0025] Use according to any one of items 1 to 4, wherein R1is -H.

[0026] Use according to any one of items 1 to 4, wherein R1is -Me. 7. Compound of general formula (II) wherein

[0027] R1’ is -H or -Me; at least one of R2’, R3’, R4’, R5’ and R6’ is a carboxylic acid substituent (COOH), preferably is one carboxylic acid in the R2’ or R6’ position; and any one of R2’, R3’, R4’, R5’ and R6’, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, - CONR7(CH2)I.6COOH, -CONR7’(CH2)I-6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i- 6SO3H;

[0028] R7’ is, if applicable, -H or -Me, preferably is -H;

[0029] R1” is -H or -Me; at least one of R2”, R3”, R4”, R5” and R6” is a carboxylic acid substituent (COOH), preferably is one carboxylic acid in the R2” or R6” position; and any one of R2”, R3”, R4”, R5” and R6”, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, - CONR7(CH2)I.6COOH, -CONR7”(CH2)I-6SO3H, -SO3H, -Cl, -F, -CF3, and - NMe2+(CH2)i-6SO3H;

[0030] R7” is, if applicable, -H or -Me, preferably is -H.

[0031] 8. Compound according to item 7, wherein the compound is a compound of formula (Ila) wherein

[0032] R1’ is -H or -Me;

[0033] R2’, R3’, R4’, and R5’ are each independently selected from the group consisting of -H, - OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, -CONR7’(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H;

[0034] R7’ is, if applicable, -H or -Me, preferably is -H;

[0035] R1” is -H or -Me;

[0036] R2”, R3”, R4”, and R5” are each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, -CONR7’(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; and

[0037] R7” is, if applicable, -H or -Me, preferably is -H.

[0038] 9. Compound according to item 7 or 8, wherein R1’ is -H.

[0039] 10. Compound according to item 7 or 8, wherein R1’ is -Me.

[0040] 11. Compound according to any one of items 7 to 10, wherein R1” is -H.

[0041] 12. Compound according to any one of items 7 to 10, wherein R1” is -Me.

[0042] 13. Compound according to item 7, wherein the compound is a compound of formula (lib) wherein each R1is independently -H or -Me.

[0043] 14. Compound according to item 7, wherein the compound is a compound of formula (He) (lie) wherein each R1is independently -H or -Me.

[0044] 15. Compound according to item 13 or 14, wherein each R1is -H.

[0045] 16. Compound according to item 13 or 14, wherein each R1is -Me.

[0046] 17. Use of a compound according to any one of items 7 to 16 as a dye.

[0047] 18. Use according to item 17, wherein the compound is used as a dye in the detection of formaldehyde or a related compound.

[0048] 19. Use according to item 17, wherein the compound is used as a dye in the detection of methanol.

[0049] 20. Process for the production of a compound of general formula (II) according to any one of items 7 to 16, the method comprising: a) reacting a compound of general formula (I) as defined in any one of items 1 to 6 with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of the compound of general formula (II); and b) optionally isolating the compound of formula (II) from the reaction.

[0050] 21. Process according to item 20, wherein the compound of general formula (II) is a compound of formula (He), and the compound of general formula (I) is a corresponding compound of formula (Ic).

[0051] 22. Process according to item 20, wherein the compound of general formula (II) is a compound as defined in item 15, and the compound of general formula (I) is a compound as defined in item 5.

[0052] 23. The process according to any one of items 20 to 22, wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof.

[0053] 24. The process according to any one of items 20 to 22, wherein the nitrogen donor is ammonia.

[0054] 25. The process according to any one of items 20 to 22, wherein the nitrogen donor is an ammonium salt.

[0055] 26. The process according to item 23 or 25, wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate.

[0056] 27. The process according to item 23 or 25, wherein the ammonium salt is ammonium acetate.

[0057] 28. The process according to any one of items 20 to 27, wherein the solvent is water.

[0058] 29. The process according to any one of items 20 to 28, wherein the reaction is carried out at a temperature ranging from 15 °C to 65 °C, such as from 20 °C to 40 °C.

[0059] 30. The process according to any one of items 20 to 29, where in the reaction is carried out at a temperature ranging from 30 °C to 40 °C.

[0060] 31 . The process according to any one of items 20 to 29, where in the reaction is carried out at 37 °C.

[0061] 32. The process according to any one of items 20 to 31 , wherein the reaction is carried out for a period of time ranging from 10 seconds to 60 minutes.

[0062] 33. The process according to any one of items 20 to 31 , wherein the reaction is carried out for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes.

[0063] 34. The process according to any one of items 20 to 31 , wherein the reaction is carried out for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

[0064] 35. Method for the detection of formaldehyde in a sample, the method comprising: a) contacting a sample with a compound of general formula (I) as defined in any one of items 1 to 6 and a nitrogen donor to form a reaction mixture and incubating the reaction mixture; and b) determining whether a compound of formula (II) as defined in any one of items 7 to 16 is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (II) is indicative of the presence of formaldehyde in the sample.

[0065] 36. Method according to item 35, wherein the compound of general formula (II) is a compound of formula (He), and the compound of general formula (I) is a corresponding compound of formula (Ic). 37. Method according to item 35, wherein the compound of general formula (II) is a compound as defined in item 15, and the compound of general formula (I) is a compound as defined in item 5.

[0066] 38. The method according to any one of items 35 to 37, wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof.

[0067] 39. The method according to any one of items 35 to 37, wherein the nitrogen donor is ammonia.

[0068] 40. The method according to any one of items 35 to 37, wherein the nitrogen donor is an ammonium salt.

[0069] 41 . The method according to item 38 or 40, wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate.

[0070] 42. The method according to item 38 or 40, wherein the ammonium salt is ammonium acetate.

[0071] 43. The method according to any one of items 35 to 42, wherein the reaction is carried out at a temperature ranging from 15 °C to 65 °C, such as from 20 °C to 40 °C.

[0072] 44. The method according to any one of items 35 to 42, wherein the reaction is carried out at a temperature ranging from 30 °C to 40 °C.

[0073] 45. The method according to any one of items 35 to 42, wherein the reaction is carried out at 37 °C.

[0074] 46. The method according to any one of items 35 to 45, wherein the reaction is carried out for a period of time ranging from 10 seconds to 60 minutes.

[0075] 47. The method according to any one of items 35 to 45, wherein the reaction is carried out for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes.

[0076] 48. The method according to any one of items 35 to 45, wherein the reaction is carried out for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

[0077] 49. Method for the detection of methanol in a sample, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to form a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (I) as defined in any one of items 1 to 6 and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; and d) determining whether a compound of formula (II) as defined in any one of items 7 to 16 is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (II) is indicative of the presence of methanol in the sample.

[0078] 50. Method according to item 49, wherein the compound of general formula (II) is a compound of formula (He), and the compound of general formula (I) is a corresponding compound of formula (Ic).

[0079] 51. Method according to item 49, wherein the compound of general formula (II) is a compound as defined in item 15, and the compound of general formula (I) is a compound as defined in item 5.

[0080] 52. The method according to any one of items 49 to 51 , wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof.

[0081] 53. The method according to any one of items 49 to 51 , wherein the nitrogen donor is ammonia.

[0082] 54. The method according to any one of items 49 to 51 , wherein the nitrogen donor is an ammonium salt.

[0083] 55. The method according to item 52 or 54, wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate.

[0084] 56. The method according to item 52 or 54, wherein the ammonium salt is ammonium acetate. 57. The method according to any one of items 49 to 56, wherein the first reaction mixture is incubated at a temperature ranging from 15 °C to 65 °C, such as from 20 °C to

[0085] 40 °C.

[0086] 58. The method according to any one of items 49 to 56, wherein the first reaction mixture is incubated at a temperature ranging from 30 °C to 40 °C.

[0087] 59. The method according to any one of items 49 to 56, wherein the first reaction mixture is incubated at 37 °C.

[0088] 60. The method according to any one of items 49 to 59, wherein the first reaction mixture is incubated for a period of time ranging from 10 seconds to 60 minutes.

[0089] 61 . The method according to any one of items 49 to 59, wherein the first reaction mixture is incubated for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes.

[0090] 62. The method according to any one of items 49 to 59, wherein the first reaction mixture is incubated for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

[0091] 63. The method according to any one of items 49 to 62, wherein the peroxide scavenging is carried out at a temperature ranging from 15 °C to 65 °C, such as from 20 °C to

[0092] 40 °C.

[0093] 64. The method according to any one of items 49 to 62, wherein the peroxide scavenging is carried out at a temperature ranging from 30 °C to 40 °C.

[0094] 65. The method according to any one of items 49 to 62, wherein the peroxide scavenging is carried out at 37 °C.

[0095] 66. The method according to any one of items 49 to 65, wherein the peroxide scavenging is carried out for a period of time ranging from 10 seconds to 60 minutes.

[0096] 67. The method according to any one of items 49 to 65, wherein the peroxide scavenging is carried out for a period of time ranging from 1 minutes to 60 minutes, such as from 10 minutes to 30 minutes.

[0097] 68. The method according to any one of items 49 to 65, wherein the peroxide scavenging is carried out for a period of time ranging from about 10 minutes to about 60 minutes, , such as from 20 minutes to 40 minutes. 69. The method according to any one of items 49 to 68, wherein the second reaction mixture is incubated at a temperature ranging from 15 °C to 65 °C, such as from 20 °C to 40 °C.

[0098] 70. The method according to any one of items 49 to 68, wherein the second reaction mixture is incubated at a temperature ranging from 30 °C to 40 °C.

[0099] 71 . The method according to any one of items 49 to 68, wherein the second reaction mixture is incubated at 37 °C.

[0100] 72. The method according to any one of items 49 to 71 , wherein the second reaction mixture is incubated for a period of time ranging from 10 seconds to 60 minutes.

[0101] 73. The method according to any one of items 49 to 71 , wherein the first reaction mixture is incubated for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes.

[0102] 74. The method according to any one of items 49 to 71 , wherein the first reaction mixture is incubated for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

[0103] 75. The method according to any one of items 35 to 74, wherein the step of determining of whether a compound of formula (II) is or has been formed comprises measuring the absorbance using a spectrophotometer, such as a UV-vis spectrophotometer, or an analyzer system comprising a spectrophotometric unit.

[0104] 76. The method according to item 75, wherein the absorbance is measured at a wavelength in the range from 350 nm to 420 nm, preferably in the range from 365 nm to 390 nm, more preferably at 378 nm.

[0105] 77. The method according to any one of items 35 to 74, wherein the step of determining of whether a compound of formula (II) is or has been formed comprises measuring visible spectrum fluorescence using a fluorometer or an analyzer system comprising a fluorometric unit.

[0106] 78. The method according to item 77, wherein the fluorescence is measured using a excitation wavelength in the range from 350 nm to 420 nm, preferably in the range from 365 nm to 390 nm, more preferably at 378 nm, and an emission / detection wavelength in the range from 420 nm to 550 nm, preferably in the range from 450 nm to 480 nm, more preferably at 470. 79. The method according to any one of items 35 to 74, wherein the compound of general formula (I) and nitrogen donor are provided together in the form of an aqueous formulation.

[0107] 80. The method according to item 79, wherein the formulation further comprises a stabilizer.

[0108] 81. The method according to item 80, wherein the stabilizer is a polar aprotic solvent, preferably Dimethyl sulfoxide (DMSO).

[0109] 82. The method according to any one of items 79 to 81 , wherein the formulation further comprises a crystallization inhibitor.

[0110] 83. The method according to item 82, wherein the crystallization inhibitor is a water- soluble polymer, preferably polyvinylpyrrolidone (PVP), more preferably PVP K90.

[0111] 84. The method according to any one of items 79 to 83, wherein the aqueous formulation has a pH ranging from 6.0 to 8.0, preferably from 6.5 to 7.5, such as about 7.0.

[0112] 85. A formulation comprising a compound of general formula (I) as defined in any one of items 1 to 6 and a nitrogen donor.

[0113] 86. The formulation according to item 85, which is a liquid, preferably aqueous, formulation.

[0114] 87. The formulation according to item 85, which is a lyophilized formulation.

[0115] 88. The formulation according to any one of items 85 to 87, wherein the compound of general formula (I) is present in a concentration ranging from 10 mg / ml to 125 mg / ml.

[0116] 89. The formulation according to item 88, wherein the compound of general formula (I) is present in a concentration ranging from 20 mg / ml to 80 mg / ml, preferably from 25 mg / ml to 75 mg / ml, more preferably from 40 mg / ml to 60 mg / ml, even more preferably from 45 mg / ml to 55 mg / ml, such as 50 mg / ml.

[0117] 90. The formulation according to any one of items 85 to 89, wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof.

[0118] 91. The formulation according to any one of items 85 to 89, wherein the nitrogen donor is ammonia.

[0119] 92. The formulation according to any one of items 85 to 89, wherein the nitrogen donor is an ammonium salt.

[0120] 93. The formulation according to item 90 or 92, wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate.

[0121] 94. The formulation according to item 90 or 92, wherein the ammonium salt is ammonium acetate.

[0122] 95. The formulation according to any one of items 92 to 94, wherein the ammonium salt is present in a concentration ranging from 0.5 M to 8 M.

[0123] 96. The formulation according to any one of items 93 to 95, wherein the ammonium salt is present in a concentration ranging from 1 M to 5 M, preferably from 1 M to 4 M, more preferably from 1.5 M to 2.5 M, such as 2 M.

[0124] 97. The formulation according to any one of items 85 to 96, further comprising a stabilizer.

[0125] 98. The formulation according to item 97, wherein the stabilizer is a polar aprotic solvent.

[0126] 99. The formulation according to item 97 or 98, wherein the stabilizer is Dimethyl sulfoxide (DMSO).

[0127] 100. The formulation according to any one of items 97 to 99, wherein the stabilizer is present in a concentration ranging from 5 % v / v to 25% v / v, preferably from 15% v / v to 25% v / v, such as 20% v / v.

[0128] 101. The formulation according to any one of items 85 to 100, further comprising an crystallization inhibitor.

[0129] 102. The formulation according to item 101 , wherein the crystallization inhibitor is a water- soluble polymer.

[0130] 103. The formulation according to item 101 or 102, wherein the crystallization inhibitor is polyvinylpyrrolidone (PVP).

[0131] 104. The formulation according to item 103, wherein the PVP is selected from the group consisting of PVP K12, PVP K25, PVP K30, PVP K60, and PVP K90.

[0132] 105. The formulation according to any one of items 102 to 104, wherein the crystallization inhibitor is PVP K90.

[0133] 106. The formulation according to any one of items 102 to 105, wherein the crystallization inhibitor is present in a concentration ranging from 0.01% w / v to 5% w / v. 107. The formulation according to any one of items 102 to 105, wherein the crystallization inhibitor is present in a concentration ranging from 0.1% w / v to 1% w / v, preferably from 0.2% w / v to 0.6% w / v, more preferably from 0.3% w / v to 0.5% w / v, such as 0.4% w / v.

[0134] 108. The formulation according to any one of items 85 to 107, wherein the formulation has a pH ranging from 6.0 to 8.5, such as from 6.5 to 7.5, such as 7.0.

[0135] 109. A kit comprising a compound of general formula (I) as defined in any one of items 1 to 6, a nitrogen donor and a reagent comprising an alcohol oxidase or alcohol dehydrogenase, and optionally a catalase.

[0136] 110. The kit according to item 109, wherein the compound of general formula (I) as defined in any one of items 1 to 6, the nitrogen donor, and the reagent comprising an alcohol oxidase or alcohol dehydrogenase are each provided in separate containers.

[0137] 111. The kit according to item 109, wherein the compound of general formula (I) as defined in any one of items 1 to 6 and the nitrogen donor are provided together in a formulation.

[0138] 112. The kit according to item 111 , wherein the formulation is the formulation according to any one of items 85 to 108.

[0139] 113. The kit according to any one of items 109 to 112, wherein the reagent is an aqueous solution.

[0140] 114. The kit according to item 113, wherein the reagent has a pH ranging from 6.0 to 8.5, such as from 7.0 to 8.0, such as 7.5.

[0141] 115. The kit according to any one of items 109 to 114, wherein the alcohol oxidase or alcohol dehydrogenase is present in a concentration ranging from 0.5 ll / rnl to 20 ll / rnl.

[0142] 116. The kit according to any one of items 109 to 114, wherein the alcohol oxidase or alcohol dehydrogenase is present in a concentration ranging from 1 ll / rnl to 10 ll / rnl, preferably from 2 ll / rnl to 8 ll / rnl, more preferably from 3 ll / rnl to 6 ll / rnl, such as 4 ll / rnl or 5 ll / rnl.

[0143] 117. The kit according to any one of items 109 to 116, wherein the catalase is present in a concentration ranging from 0.05 kll / ml to 5 kll / ml.

[0144] 118. The kit according to any one of items 109 to 116, wherein the catalase is present in a concentration ranging from 0.5 kll / ml to 2.5 kU / ml, preferably from 0.5 kU / ml to 1.5 kU / ml, such as 1 kU / ml. 119. Use of a formulation according to any one of items 85 to 108 for detecting formaldehyde in a sample.

[0145] 120. Use of a kit according to any one of items 109 to 118 for detecting methanol in a sample.

[0146] Detailed description of the invention

[0147] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of chemistry and diagnostics.

[0148] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0149] The original Hantzsch pyridine synthesis or Hantzsch dihydropyridine synthesis is a multicomponent organic reaction between an aldehyde such as formaldehyde, 2 equivalents of a P-keto ester such as ethyl acetoacetate and a nitrogen donor such as ammonium acetate or ammonia. The initial reaction product is a dihydropyridine which can be oxidized in a subsequent step to a pyridine. The driving force for this second reaction step is aromatization. This reaction was reported by Hantzsch, A. (1881) Chemische Berichte 14(2) 1637-1638. A more recent discussion can be found in “Name Reactions, A Collection of Detailed Mechanisms and Synthetic Applications”, 3rd ed. (2006) by Jie Jack Li, “Hantzsch dihydropyridine synthesis”, pp 281-282. Although initially described with p-ketoesters in the original paper of Arthur Hantzsch, there are several reports on analogous reactions with other carbonyl compounds such as p-ketoamides and p-diketones. In addition, there are reports on using Hantzsch-type reactions for derivatization of target compounds, e.g. prior to HPLC- based analytics.

[0150] Example 1 describes the screening of several p-ketocarbonyl compounds. To this end, the inventors first focused on formaldehyde detection by the Hantzsch reaction (i.e. Hantzsch dihydropyridine synthesis) as such. The compounds considered were selected from the group consisting of acetoacetamide (AA), N-(acetoacetyl)anthranilic acid (N-AAAA), potassium N- (acetoacetyl)sulfanilate (K-AAsA), and acetylacetone (AA).

[0151] In making the selection, the inventors excluded compounds such as salts of p-ketoacids or p- ketoesters due to their instable nature particularly in a Hantzsch formaldehyde detection reagent containing high concentrations of ammonia and ammonium. The inventors also found that no useful results in formaldehyde detection were obtained when using 1 ,3- cyclohexanedione with ammonium donors as described by Sawicki and Carnes [doi: 10.1007 / BF01216118] (data not shown), which we omitted this p-ketocarbonyl early in our studies. The use of / \ / -(acetoacetyl)anilide required a significant amount of an organic solvent which is why we focussed on water-soluble candidates for the formaldehyde derivatization. The panel consisted of the p-ketocarbonyls acetoacetamide (AA), N-(acetoacetyl)anthranilic acid (N-AAAA), potassium N-(acetoacetyl)sulfanilate (K-AAsA) and acetylacetone (AA). An enzyme-empty aqueous buffered solution reflecting, was mixed with the Hantzsch formaldehyde-derivatization reagent (containing one of the four p-ketocarbonyl candidates) and aqueous formaldehyde solutions (0-1 g / L) were added and the formation of the dihydropyridine was followed by absorbance readout at 378 and 405 nm (Figure 1).

[0152] To screen for a suitable p-ketocarbonyl compound, the inventors concentrated directly on formaldehyde detection by the Hantzsch reaction (or more exact Hantzsch dihydropyridine synthesis) in water or in a more complex aqueous mixture, e.g. a mixture containing ingredients of biological origin. That is to say, the initial experiments did not take into consideration formaldehyde analyte as a transformative product, e.g. from a chemical or enzymatic reaction which would release formaldehyde.

[0153] In their selection of candidate Hantzsch reactants, the inventors excluded compounds such as salts of p-ketoacids or p-ketoesters due to their instable nature, particularly in the presence of high concentrations of ammonia and ammonium. Furthermore, preliminary results failed to indicate promising results in formaldehyde detection in using 1 ,3-cyclohexanedione with ammonium donors as described by Sawicki E. and Carnes R.A. Microchim Acta 1 (1968) 148- 159. The use of / \ / -(acetoacetyl)anilide required a significant amount of an organic solvent which is why the inventors concentrated on water-soluble candidates for the formaldehyde derivatization. The panel consisted of the p-ketocarbonyls acetoacetamide (AA), N-(acetoacetyl)anthranilic acid (N-AAAA), potassium N-(acetoacetyl)sulfanilate (K-AAsA) and acetylacetone (AA). An buffer with a complex composition (emulating a sample with components of biological origin) or water (instead of the buffer) was mixed with the Hantzsch formaldehyde-derivatization reagent (containing one of the four p-ketocarbonyl candidates). Aqueous formaldehyde solutions with pre-defined concentrations (0-1 g / L) were added and the formation of the dihydropyridine was followed by absorbance readout at 378 and 405 nm.

[0154] At pH 6.8 of the respective reagent in the screening, N-AAAA demonstrated several advantages over other p-ketocarbonyl compounds, including superior kinetics / saturation, extinction coefficients, and wavelength independence. Unlike, the p-ketoamides, AA exhibited a better detectability at 405 nm, achieving the strongest absorption and signal when it was comprised in a slightly acidic reagent. Conversely, AAA showed good kinetics in terms of reaching saturation, despite having the least favorable extinction coefficients. The wavelength difference was consistent with literature. Dihydropyridines formed from p-ketoamides or p- ketoesters were best detected spectrophotometrically at around 375 nm, whereas the dihydropyridines from p-ketoketones were better detected at slightly red-shifted wavelengths around 410 nm (Li, Q. et al. Anal Chim Acta 612 (2008) 165-172 and Nash, T. Biochem J 55 (1953) 416-421).

[0155] At pH 7.8 as opposed to pH 6.8, the reactions proceeded more rapidly and exhibited higher extinction values, with the exception of AA. The pH-dependency of Hantzsch reactions with different p-ketocarbonyl compounds that the inventors observed is known from the literature. AA generally requires an acidic environment (Nash, T. supra), while for - ketoacetamides a slightly alkaline pH is preferred (Li, Q. supra). In the analyses of the present study, N-AAAA consistently showed advantages over other p-ketocarbonyl compounds, including superior kinetics / saturation, extinction coefficients, a broader wavelength range. While AA remained more detectable at 405 nm, it was still not as effective as N-AAAA at this pH level. AAA, despite having the least favorable extinction coefficients at the wavelengths tested, exhibited the best kinetics, showing apparent saturation of the reaction after 20 min.

[0156] In conclusion, N-AAAA consistently outperforms other p-ketocarbonyl compounds when used as a Hantzsch reagent at pH levels compatible with a formaldehyde detection reaction. This makes it the most suitable candidate for formaldehyde detection in our assays. Its superior kinetics, saturation properties, and wavelength independence render it highly effective, ensuring reliable and robust measurement outcomes.

[0157] The inventors further found that formulations of higher than 15-20 mg / mL of K-AAsA in 4 M of Ammonium acetate with slightly alkaline pH crystallized quickly when stored at refrigerated temperatures.

[0158] Candidate screened reagents using AA as the p-ketocarbonyl displayed the highest reactivity when present in a slightly acidic reagent containing a suitable ammonium donor. Such reagents are however known for their short shelf life and tendency to form absorbing byproducts progressively, as disclosed in WO2015073275A1. Compared to the classical reagent according to Nash, T. (supra) which is made from AA, ammonium acetate, and acetic acid, the inventors also tried Hantzsch reagents with diammonium hydrogen phosphate and ammonium dihydrogen phosphate as ammonium donors, instead of ammonium acetate, suspecting better stability and similar kinetics. Although reasonably stable in the short term (4 weeks at room temperature), the inventors could not further stabilize such Nash-like reagent with chelators (EDTA, citric acid) or antioxidants (ascorbic acid) for long-term storage (over 12 months) under refrigerated conditions.

[0159] AA showed only a low signal response in the formaldehyde detection assay, which is why the inventors decided against further use.

[0160] As noted above, the present inventors have identified p-keto amide compounds as superior and sufficiently chemoselective detection reagent in the detection of formaldehyde and related compounds, such as methanol. These p-keto amide compounds are collectively referred to herein as compound(s) of general formula (I), of which N-(acetoacetyl)anthranilic acid is a particularly preferred example. The compound(s) of general formula (I) when used as a Hantzsch reagent has shown to be superior over other p-ketocarbonyl compounds because of its unusually high solubility in aqueous solutions (higher than 10 % w / w), superior kinetics and a high signal in the corresponding reaction with formaldehyde. In addition, the compound(s) of general formula (I) displayed a good stability when compared with other representatives of the classes.

[0161] The present invention thus provides in a first aspect the use of a compound of general formula (I) or a salt thereof in a Hantzsch reaction wherein

[0162] R1is -H or -Me; at least one of R2, R3, R4, R5and R6is a carboxylic acid substituent ( COOH), preferably is one carboxylic acid in the R2or R6position; and any one of R2, R3, R4, R5and R6, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, - OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7(CH2)I.6COOH, -CONR7(CH2)I.6SO3H, - SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; and

[0163] R7is, if applicable, -H or -Me, preferably is -H.

[0164] In some embodiments, the compound of general formula (I) is a compound of formula (la) wherein

[0165] R1is -H or -Me; R2, R3, R4, and R5are each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7(CH2)I.6COOH, -CONR7(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; and

[0166] R7is, if applicable, -H or -Me, preferably is -H.

[0167] In some embodiments, R1in is -H.

[0168] In some embodiments, R1is -Me.

[0169] In some embodiments, wherein the compound of general formula (I) is a compound of formula (lb) wherein R1is -H or -Me.

[0170] In some embodiments, R1in is -H.

[0171] In some embodiments, R1is -Me.

[0172] In some embodiments, the compound of general formula (I) is a compound of formula (Ic) wherein R1is -H or -Me.

[0173] In some embodiments, R1is -H.

[0174] In some embodiments, R1is -Me.

[0175] A compound of general formula (I) when reacted with formaldehyde in the presence of a nitrogen donor, such as ammonia or an ammonium salt, results in the production of a dihydropyridine. The resulting dihydropyridine, which is collectively referred to herein as compound(s) of general formula (II), can then be detected, e.g. spectrophotometrically, and thus may server as a suitable dye in the detection of formaldehyde and related compounds, such as methanol.

[0176] The present invention thus provides in a further aspect a compound of general formula (II) wherein

[0177] R1’ is -H or -Me; at least one of R2’, R3’, R4’, R5’ and R6’ is a carboxylic acid substituent (COOH), preferably is one carboxylic acid in the R2’ or R6’ position; and any one of R2’, R3’, R4’, R5’ and R6’, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, -CONR7’(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H;

[0178] R7’ is, if applicable, -H or -Me, preferably is -H;

[0179] R1” is -H or -Me; at least one of R2”, R3”, R4”, R5” and R6” is a carboxylic acid substituent (COOH), preferably is one carboxylic acid in the R2” or R6” position; and any one of R2”, R3”, R4”, R5” and R6”, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7”(CH2)I.6COOH, -CONR7”(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H;

[0180] R7” is, if applicable, -H or -Me, preferably is -H.

[0181] In some embodiments, R1’ is -H.

[0182] In some embodiments, R1” is -H.

[0183] In some embodiments, R1’ is -H and R1” is -Me.

[0184] In some embodiments, R1’ is -Me and R1” is -H.

[0185] In some embodiments, R1’ and R1” are each -H.

[0186] In some embodiments, R1’ and R1” are each -Me.

[0187] In some embodiments, R2’ is COOH. In some embodiments, R6’ is COOH.

[0188] In some embodiments, R2” is COOH.

[0189] In some embodiments, R6” is COOH.

[0190] In some embodiments, R2’ and R2” are each COOH.

[0191] In some embodiments, R6’ and R6” are each COOH.

[0192] In some embodiments, each of R2’, R3’, R4’ and R5’ is -H, and R6’ is COOH.

[0193] In some embodiments, each of R2”, R3”, R4” and R5” is -H, and R6” is COOH.

[0194] In some embodiments, each of R2’, R3’, R4’ and R5’ is -H, and R6’ is COOH, and each of R2”, R3”, R4” and R5” is -H, and R6” is COOH.

[0195] In some embodiments, the compound of general formula (II) is a compound of formula (Ila) wherein

[0196] R1’ is -H or -Me;

[0197] R2’, R3’, R4’, and R5’ are each independently selected from the group consisting of -H, -OH, - Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, - CONR7’(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H;

[0198] R7’ is, if applicable, -H or -Me, preferably is -H;

[0199] R1” is -H or -Me;

[0200] R2”, R3”, R4”, and R5” are each independently selected from the group consisting of -H, -OH, - Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, - CONR7’(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; and

[0201] R7” is, if applicable, -H or -Me, preferably is -H.

[0202] In some embodiments, R1’ is -H.

[0203] In some embodiments, R1” is -H. some embodiments, R1’ is -H and R1” is -Me.

[0204] In some embodiments, R1is -Me and R1is -H.

[0205] In some embodiments, R1’ and R1” are each -H.

[0206] In some embodiments, R1’ and R1” are each -Me.

[0207] In some embodiments, each of R2’, R3’, R4’ and R5’ is -H.

[0208] In some embodiments, each of R2”, R3”, R4” and R5” is -H.

[0209] In some embodiments, each of R2’, R3’, R4’ and R5’ is -H, and each of R2”, R3”, R4” and R5” is - H.

[0210] In some embodiments, the compound of general formula (II) is a compound of formula (lib) wherein each R1is independently -H or -Me.

[0211] In some embodiments, each R1is -H.

[0212] In some embodiments, each R1is -Me.

[0213] In some embodiments, the compound of general formula (II) is a compound of formula (He) wherein each R1is independently -H or -Me.

[0214] In some embodiments, each R1is -H.

[0215] In some embodiments, each R1is -Me. As noted above, compound(s) of general formula (II) can then be detected, e.g. spectrophotometrically, and thus may server as a suitable dye in the detection of formaldehyde and related compounds, such as methanol.

[0216] The present invention thus provides in a further aspect the use of a compound of general formula (II) as defined herein as a dye.

[0217] In some embodiments, the compound of general formula (II) is used as a dye in the detection of formaldehyde or a related compound.

[0218] In some embodiments, the compound of general formula (II) is used as a dye in the detection of methanol.

[0219] As noted above, compound(s) of general formula (II) is a dihydropyridine obtainable by using a p-keto amide compound of general formula (I) as a reactant in Hantzsch pyridine synthesis (also referred to herein as Hantzsch reaction). Generally, in the Hantzsch pyridine synthesis a compound of general formula (I) is reacted with formaldehyde in the presence of a nitrogen donor, such as ammonia or an ammonium salt, to form a compound of general formula (II). Conditions suitable for driving the Hantzsch pyridine synthesis are well known by the skilled person, and are equally applicable in the present invention.

[0220] The present invention thus provides in a further aspect a process for the production of a compound of general formula (II), the method comprising: a) reacting a compound of general formula (I) as defined herein with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of the compound of general formula (II); and b) optionally isolating the compound of formula (II) from the reaction.

[0221] In some embodiment, the process comprises reacting a compound of general formula (la) as defined herein with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of the compound of general formula (II), and in particular a compound of formula (Ila).

[0222] In some embodiment, the process comprises reacting a compound of general formula (lb) as defined herein with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of the compound of general formula (II), and in particular a compound of formula (lib).

[0223] In some embodiment, the process comprises reacting a compound of general formula (Ic) as defined herein with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of the compound of general formula (II), and in particular a compound of formula (He). In one embodiment, the process comprises reacting a compound of general formula (Ic) wherein R1is -H, with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of a compound of general formula (lie) wherein each R1is -H.

[0224] The nitrogen donor may be any suitable nitrogen donor, which can be used in the Hantzsch pyridine synthesis. Suitable nitrogen donors are well know to the skilled person. Non-limiting examples of a nitrogen donor, which can be used in the process of the present invention, are ammonia and ammonium salts, such as ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate.

[0225] Accordingly, in some embodiments, the nitrogen donor is ammonia.

[0226] In some embodiments, the nitrogen donor is an ammonium salt.

[0227] In some embodiments, the nitrogen donor is an ammonium salt selected from the group consisting of ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate and diammonium sulfate.

[0228] In some embodiments, the nitrogen donor is ammonium acetate. The solvent may be any suitable solvent which can be used in the Hantzsch pyridine synthesis. Suitable solvents are well known to the skilled person. A non-limiting example of a solvent is water.

[0229] As noted above, conditions suitable for driving the Hantzsch pyridine synthesis are well known by the skilled person (as described in, e.g., by Jie Jack Li, “Hantzsch dihydropyridine synthesis”, pp 281-282 in: “Name Reactions, A Collection of Detailed Mechanisms and Synthetic Applications”, 3rd ed., 2006), and are equally applicable in the present invention. Generally, the reaction can be carried out at a temperature ranging from 15 °C to 65 °C, and for a period of time ranging from 10 seconds to 60 minutes. Yet, it is also possible to carry out a preparative dihydropyridine synthesis under reflux (-100 °C with water) or in sealed vessels with microwave radiation.

[0230] In some embodiments, the reaction is carried out for at a temperature ranging from 15 °C to 65 °C.

[0231] In some embodiments, the reaction is carried out at a temperature ranging from 20 °C to 40 °C.

[0232] In some embodiments, the reaction is carried out at a temperature ranging from 30 °C to 40 °C.

[0233] In some embodiments, the reaction is carried out at 37 °C.

[0234] In some embodiments, the reaction is carried out for a period of time ranging from 10 seconds to 60 minutes.

[0235] In some embodiments, the reaction is carried out for a period of time ranging from 1 minute to 60 minutes.

[0236] In some embodiments, the reaction is carried out for a period of time ranging from 10 minutes to 60 minutes.

[0237] In some embodiments, the reaction is carried out for a period of time ranging from 10 minutes to 30 minutes.

[0238] In some embodiments, the reaction is carried out for a period of time ranging from 20 minutes to 40 minutes.

[0239] The present inventors have also found that the formation of the dihydropyridine resulting from the reaction of the p-keto amide compound of general formula (I) and formaldehyde in the present of the nitrogen donor can be suitably used as a measure of the presence of formaldehyde in a sample. As noted above, compound(s) of general formula (II) can be detected, e.g. spectrophotometrically, and thus may server as a suitable dye in the detection of formaldehyde and related compounds, such as methanol. The present inventors have thus further developed a detection method which makes use of the principles of the pyridine synthesis, and the detectability of the resulting dihydropyridine.

[0240] The present invention thus provides in a further aspect a method for the detection of formaldehyde in a sample, the method comprising: a) contacting a sample with a compound of general formula (I) as defined herein and a nitrogen donor to form a reaction mixture and incubating the reaction mixture; and b) determining whether a compound of formula (II) as defined herein is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (II) is indicative of the presence of formaldehyde in the sample.

[0241] In some embodiment, the method comprises a) contacting a sample with a compound of general formula (la) as defined herein a nitrogen donor to from a reaction mixture and incubating the reaction mixture; and b) determining whether a compound of formula (Ila) as defined herein is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (Ila) is indicative of the presence of formaldehyde in the sample.

[0242] In some embodiment, the method comprises a) contacting a sample with a compound of general formula (lb) as defined herein a nitrogen donor to form a reaction mixture and incubating the reaction mixture; and b) determining whether a compound of formula (lib) as defined herein is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (lib) is indicative of the presence of formaldehyde in the sample.

[0243] In some embodiment, the method comprises a) contacting a sample with a compound of general formula (Ic) as defined herein a nitrogen donor to form a reaction mixture and incubating the reaction mixture; and b) determining whether a compound of formula (He) as defined herein is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (lie) is indicative of the presence of formaldehyde in the sample.

[0244] In one embodiment, the method comprises a) contacting a sample with a compound of general formula (Ic) a nitrogen donor to form a reaction mixture and incubating the reaction mixture wherein R1is -H; and determining whether a compound of formula (He) wherein each R1is -H, is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (He) is indicative of the presence of formaldehyde in the sample.

[0245] Since the principal reaction underlying the detection method is the same as that described above for the production of a dihydropyridine of general formula (II), i.e. it is a Hantzsch pyridine synthesis, the above described conditions and details, notably those given with respect to the nitrogen donor, temperature and duration, apply mutatis mutandis to the detection method, and more specifically to step a) thereof.

[0246] Formaldehyde is a common intermediate product formed in the metabolization of various related compounds, such as methanol. Hence, the formation and detection of a dihydropyridine of general formula (II) resulting from a Hantzsch reaction of formaldehyde and a p-keto amide compound of general formula (I), can also be applied to methods of detecting theses related compounds. In such cases, the method may require some pre-treatment step by which the compound of interest is metabolized to formaldehyde. Such pre-treatment step may involve the use of one or more enzymes, which are known to be involved in the metabolization of the compound of interest to formaldehyde. Such pre-treatment step is however not limited to enzymatic reactions, but may also involve chemical reactions which result in the production of formaldehyde.

[0247] A compound of particular interest is methanol. By way of example, methylotrophic yeasts (such as Pichia pastoris, Komagataella phaffii, Hansenula polymorpha, or Ogataea polymorpha) are workhorses in high yield recombinant protein production processes. Methanol is used to induce and maintain protein expression during the fermentation. The methanol content in the medium during the fermentation needs to be tightly controlled to balance maximum productivity and minimal methanol toxicity. Thus, knowing the exact content of methanol in the fermentation broth is of great importance.

[0248] In Example 2 the inventors further evaluated the compounds N-AAAA, AA, AAA and K-AAsA in a setting wherein formaldehyde was generated by oxidation of methanol by way of enzymatic reaction catalyzed by an alcohol oxidase. Thus, the step of formaldehyde detection using the Hantzsch dihydropyridine reaction was conducted directly in the enzyme reaction mixture, i.e. in parallel while formaldehyde was generated (one-pot reaction), and not sequentially in two subsequent separate reactions. Measurements were taken on the Cedex® Bio HT analyzer. The results are shown in Figure 2 and Table 1. The reagents for alcohol oxidation and the formaldehyde detection mediated by the Hantzsch dihydropyridine synthesis were mixed before the sample (0-5 g / L of methanol solutions in water) was added.

[0249] Across the different pH values of the added Hantzsch dihydropyridine synthesis reagent, the use of reagent N-AAAA stood out prominently. It exhibited the highest signal and the steepest slope of signal increase at both detection wavelengths. The p-ketoamides were generally detectable better at 378 nm, while the p-ketoketone AA performed better at 409. N-AAAA demonstrated the best performance across all tested conditions compared to the other p- ketocarbonyl compounds. Furthermore, there was no preference for AA at a lower pH in the one-pot reaction here, as opposed to what was initially found for formaldehyde alone, as shown in Example 1. This suggests that the biocompatibility of a Hantzsch reagent consisting of N- AAAA and ammonium acetate with respect to the enzymes catalase and alcohol oxidase was superior. Even at 409 nm and under slightly more acidic conditions, AA did not match the performance of N-AAAA. It was also noted that the alcohol oxidase used had an optimal pH of about 7.5 which could at least partially explain this finding. Table 1 : Absorbance differences, indicated as “AA”. Differences are calculated from the absorbance after ~ 9 min on the one hand, and the absorbance directly after sample addition on the other hand.

[0250] Thus, N-AAAA consistently exhibited the best formaldehyde-detection performance among all tested p-ketocarbonyl compounds in a one-pot reaction with simultaneous methanol oxidation. This includes aspects such as kinetics, signal strength, wavelength independence as well as biocompatibility.

[0251] While the results indicate that even a single reagent mixture containing all components for methanol detection, including the Hantzsch reactants, provide workable embodiments for methanol detection, the inventors the inventors also contemplate sequential reactions. These apply not only to methanol assays but to any other reactions (enzymatic or chemically, or just in the form of extraction). The skilled person will make selections in this regard, depending specific requirement.

[0252] Example 3 provides further details on a methanol detection assays based on N-AAAA and conditions for enzymatic oxidation of methanol and subsequent formaldehyde detection. The inventors directed further experimentation to the composition of the Hantzsch reagents with N- AAAA (HR) to fine-tune stability and compatibility with the Roche Cedex® analyzers, and to the alignment with the measurement, device and storage requirements.

[0253] The inventors and others have noted a stability decrease when handling Hantzsch reagents that can be attributed to the formation of byproducts leading to coloring and absorbance increase in the detection wavelength range. Increasing background signals impede the quantification of formaldehyde when not corrected in the quantification, or they deplete the available reaction equivalents to generate signal. For the beta-ketoacetamides, the inventors found a second stability-determining factor. Crystallization of by-products of the parent compound was a symptom of aminolysis and subsequent aldol-type reactions as was found using LC / MS analytics. The formation of these by-products does not impact the Hantzsch reaction in general, but decreases the available beta-ketoacetamides during the reaction. In addition, the sparingly soluble components introduce noise in the cuvette which is why conditions were sought which allowed to prolong shelf life or storage time.

[0254] Initially, the inventors tried different basic buffers and additives in the Hantzsch reagent, with ammonium phosphate buffer as the ammonia donor. Ammonium phosphate buffer held good promises in stability but ammonium acetate led to better kinetics.

[0255] To optimize the Hantzsch reagent for the formaldehyde derivatization via the Hantzsch dihydropyridine synthesis, the inventors found it advantageous to select 50 mg / mL of N- (acetoacetyl)anthranilic acid in 4 M of ammonium acetate buffer as a starting point formulation (formulation Ob), the inventors then varied the pH, buffer concentration, buffer type and several additives.

[0256] Different embodiments of Hantzsch reagents disclosed in Example 3 were tested, using a sequential setting on the Cedex® Bio HT with separated alcohol oxidase reagent (R1) and Hantzsch reagents (HR). Monitoring was directed to the reagent background AA1 and dihydropyridine signal AA2 at different time points under accelerated temperature storage conditions at 35 °C over several weeks. Additionally, there was monitored the time until the crystallization of the by-product in the reagents set in. AA1 and AA2 in dependency of the incubation time were fitted by a linear model to obtain slopes for comparison. The data are depicted in Table 2.

[0257] Table 2: Performance data of different HR formulations (0a, Ob, 1 , 2, 4-1 , 4-2, 4-3, 4-4, 4-5, 4- 6, 4-7, 4-8, 5, 6, 7, 9, 10, 11 , 12, composition see Example 3) in a methanol-detection assay (formaldehyde detection via HR, each HR contains beta-ketocarbonyls and ammonium source for the Hantzsch dihydropyridine synthesis). Slopes have been determined by linear fitting of the background increase (AA1) over time and methanol signal (AA2) over over accelerated storage time at 35 °C. BNIB = bigger number is better, LNIB = lower number is better. N.d. = not determined.

[0258] DMSO, PVP, a more alkaline pH, lower ammonia concentration and ammonium phosphate enhance stability in that the tendency for crystallization is lowered. Notably, the starting point HR formulation 0a shows 10 d without crystallization at 35 °C (accelerated storage stress) but 8-9 months without crystallization at the recommended storage temperature (2-8 °C) showing that any improvement pushes the goal toward the storage time of about 1.5 years at 2-8 °C. Background absorbance increase (AA1) is detectable at higher pH, higher ammonium concentration, as well as DMSO and the presence of PVP, but not for all PVP / DMSO combinations. The presence of other organic additives slightly decreases AA1 stability, but a high pH and a high ammonium concentration is the main driver for instability. AA2 determines the amount of signal in the methanol detection. Generally, the higher the pH and the higher the ammonium concentration, the higher AA2. PVP and DMSO can enhance AA2 further. A higher pH leads to a faster Hantzsch dihydropyridine reaction in line with the experiments shown in Examples 1 and Example 2.

[0259] The present invention thus provides in a further aspect a method for the detection of methanol in a sample, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to from a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (I) as defined herein and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; d) determining whether a compound of formula (II) as defined herein is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (II) is indicative of the presence of methanol in the sample.

[0260] In some embodiments, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to from a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (la) as defined herein and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; d) determining whether a compound of formula (Ila) as defined herein is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (Ila) is indicative of the presence of methanol in the sample.

[0261] In some embodiments, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to from a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (lb) as defined herein and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; d) determining whether a compound of formula (lib) as defined herein is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (lib) is indicative of the presence of methanol in the sample. In some embodiments, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to from a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (Ic) as defined herein and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; d) determining whether a compound of formula (He) as defined herein is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (He) is indicative of the presence of methanol in the sample.

[0262] In some embodiments, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to from a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (Ic) and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; wherein R1is -H; and d) determining whether a compound of formula (He) wherein each R1is -H, is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (Ila) is indicative of the presence of methanol in the sample.

[0263] The first reaction mixture can be incubated at any suitable temperature which allows the conversion of methanol to formaldehyde by the alcohol oxidase or alcohol dehydrogenase, and it usually dependents on the optimum temperature of the particular enzyme(s) applied. Any kind of alcohol oxidase or alcohol dehydrogenase can be used in the present invention. The enzyme may thereby be a recombinantly produced enzyme, an enzyme isolated from a naturally occurring source, or may be chemically synthesized. Generally, the first reaction mixture can be incubated at a temperature ranging from 15 °C to 65 °C, and for a period of time ranging from 10 seconds to 60 minutes.

[0264] In some embodiments, the first reaction mixture is incubated at a temperature ranging from 15 °C to 65 °C.

[0265] In some embodiments, the first reaction mixture is incubated at a temperature ranging from 20 °C to 40 °C.

[0266] In some embodiments, the first reaction mixture is incubated at a temperature ranging from 30 °C to 40 °C.

[0267] In some embodiments, the first reaction mixture is incubated at 37 °C.

[0268] In some embodiments, the first reaction mixture is incubated for a period of time ranging from 10 seconds to 60 minutes.

[0269] In some embodiments, the first reaction mixture is incubated for a period of time ranging from 1 minute to 60 minutes.

[0270] In some embodiments, the first reaction mixture is incubated for a period of time ranging from 10 minutes to 60 minutes. In some embodiments, the first reaction mixture is incubated for a period of time ranging from 10 minutes to 30 minutes.

[0271] In some embodiments, the first reaction mixture is incubated for a period of time ranging from 20 minutes to 40 minutes.

[0272] Although entirely optional, including a peroxide scavenging step has the benefit of removing unwanted amounts of peroxide formed during the conversion of methanol to formaldehyde, which can "poison" the enzyme alcohol oxidase, thereby making the enzyme solution more stable. Additionally, this step helps to remove peroxide from the equilibrium. The catalase regenerates half an equivalent of molecular oxygen, which is needed again in the oxidation step. Furthermore, the peroxide may react with components of the reaction mixture or sample (such as proteins or small molecules), which could potentially generate a background signal (absorbance or turbidity), but does not necessarily have to.

[0273] The peroxide scavenging can be carried our at any suitable temperature which allows the conversion of peroxide to H2O and O2 by the catalase, and it usually dependents on the optimum temperature of the particular enzyme applied. Any kind of catalase can be used in the present invention. The catalase may thereby be a recombinantly produced enzyme, an enzyme isolated from a naturally occurring source, or may be chemically synthesized. Generally, the peroxide scavenging can be carried out at a temperature ranging from 15 °C to 65 °C, and for a period of time ranging from 10 seconds to 60 minutes.

[0274] In some embodiments, the peroxide scavenging is carried out at a temperature ranging from 15 °C to 65 °C.

[0275] In some embodiments, the peroxide scavenging is carried out at a temperature ranging from 20 °C to 40 °C.

[0276] In some embodiments, the peroxide scavenging is carried out at a temperature ranging from 30 °C to 40 °C.

[0277] In some embodiments, the peroxide scavenging is carried out at 37 °C.

[0278] In some embodiments, the peroxide scavenging is carried out for a period of time ranging from 10 seconds to 60 minutes.

[0279] In some embodiments, the peroxide scavenging is carried out for a period of time ranging from 1 minutes to 60 minutes.

[0280] In some embodiments, the peroxide scavenging is carried out for a period of time ranging from 10 minutes to about 60 minutes. In some embodiments, the peroxide scavenging is carried out for a period of time ranging from, such as from 10 minutes to 30 minutes.

[0281] In some embodiments, the peroxide scavenging is carried out for a period of time ranging from 20 minutes to 40 minutes.

[0282] Since the principal reaction underlying the detection method is the same as that described above for the production of a dihydropyridine of general formula (II), i.e. it is a Hantzsch pyridine synthesis, the above described conditions and details, notably those given with respect to the nitrogen donor, temperature and duration, apply mutatis mutandis to the detection method, and more specifically to the second reaction as defined in step c) thereof.

[0283] The formation of the dihydropyridine of general formula (II) in any of the above-described detection methods may be determined using any suitable means well-know to the skilled person. The dihydropyridine may for example be determined spectrophotometrically or fluorometrically using appropriate detection devises.

[0284] Thus, the step of determining of whether a compound of formula (II) is or has been formed may comprise measuring the absorbance using a spectrophotometer, such as a UV-vis spectrophotometer, or an analyzer system comprising a spectrophotometric unit. Non-limiting examples of such an analyzer system are the Cedex® Bio or Cedex® Bio HT analyzers, which are commercially available from Roche Diagnostics. Suitably, the absorbance may be measured at a wavelength in the range from 350 nm to 420 nm, preferably in the range from 365 nm to 390 nm, more preferably at 378 nm.

[0285] Alternatively, the step of determining of whether a compound of formula (II) is or has been formed may comprise measuring visible spectrum fluorescence using a fluorometer or an analyzer system comprising a fluorometric unit. Non-limiting examples of such an analyzer system are Agilent BioTek Synergy™ Microplate reader, Agilent Cary Eclipse fluorescence spectrometer, TECAN Spark® microplate reader, TECAN Infinite® reader, and Promega GloMax® Explorer. Suitable, the fluorescence is measured using an excitation wavelength in the range from 350 nm to 420 nm, preferably in the range from 365 nm to 390 nm, more preferably at 378 nm, and an emission / detection wavelength in the range from 420 nm to 550 nm, preferably in the range from 450 nm to 480 nm, more preferably at 470.

[0286] In the detection methods of the present invention, the compound of general formula (I) and the nitrogen donor may be added to the sample either simultaneously, e.g., in form of a formulation containing both, or sequentially.

[0287] In some embodiments, compound of general formula (I) and the nitrogen donor are brough in contact with the sample (e.g., added to the sample) in form of an aqueous formulation containing both. In some embodiments, the aqueous formulation is a formulation described in more detail below.

[0288] Various types of samples may be used in detection methods of the present invention.

[0289] Preferably, the sample is in a liquid form, such as an aqueous solution or suspension.

[0290] The sample may comprise a biological sample. The sample may comprise, e.g., a clinical sample (i.e. , a sample collected in a clinical or veterinary setting, e.g., by or at the request or supervision or direction of a doctor, nurse, aid worker, or medic) and / or a physiological sample (a sample collected from an organism, e.g., a mammal such as a human). In some embodiments, the biological sample comprises a biological fluid. Non-limiting examples of biological fluids include sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, and fluid obtained from plants such as the fluid transported in xylem cells or phloem sieve tube elements of a plant (e.g. sap). The sample may be a liquid solution or suspension or composition comprising cultured cells or tissue or a supernatant of such a solution or suspension, e.g., a sample of conditioned media or a sample of growth media in which a population of cells was cultured. In some embodiments, the sample is within a culture (e.g., inserted into a bioreactor) or provided from a media, culture, or reaction, e.g., in a bioreactor. For example, the sample may be within or provided from a fermenter such as a culture or culture supernatant from a fermentation reaction (e.g., an ongoing fermentation, such as during beer / wine production, the culture of cells in research settings, the production of a compound, etc.). The sample may comprise a beverage or food composition, preferably beverage or a fluid obtained from a food composition. In some embodiments, the beverage or food composition comprises a fermented composition. A food composition may comprise, e.g., a plant or a composition isolated from a plant, and / or an animal or a composition isolated from an animal. In various embodiments, a sample comprises a beverage composition. Non-limiting examples of beverage compositions include soft drinks, fountain beverages, water, coffee, tea, milk, dairy-based beverages, soy-based beverages (e.g., soy milk), almond-based beverages (e.g., almond milk), vegetable juice, fruit juice, fruit juice-flavored drinks, energy drinks, sports and fitness drinks, alcoholic products, and beverages comprising any combination thereof. Non-limiting examples of beverage compositions comprising water include purified water (e.g., filtered water, distilled water, or water purified by reverse osmosis), flavored water, mineral water, spring water, sparkling water, tonic water, and any combination thereof. In various embodiments, the sample comprises alcohol. Non-limiting examples of such samples include samples comprising or obtained / provided from beer, malt beverages, liqueur, wine, spirits, and any combination thereof. The sample may comprise an environmental fluid, such as marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, and ground water.

[0291] The present invention provides in a further aspect, a formulation comprising a compound of general formula (I) as defined herein and a nitrogen donor.

[0292] The formulation may be in any suitable form. The formulation may be a liquid, preferably aqueous, formulation. It may also be a powder or lyophilized formulation. Preferably, the formulation is an aqueous formulation. An aqueous formulation according to present invention may be prepared by adding the components in their dried (powdered) form to a suitable aqueous solvent, such as water. The aqueous formulation may also be prepared by reconstituting a lyophilized formulation in, e.g. water or any other suitable aqueous solvent.

[0293] The concentration of the compound of general formula (I) in the formulation is not particularly limited, and may be adapted as the need requires. Generally, the compound of general formula (I) may be present in a concentration ranging from 10 mg / ml to 125 mg / ml.

[0294] In some embodiments, the compound of general formula (I) is present in a concentration ranging from 20 mg / ml to 80 mg / ml, preferably from 25 mg / ml to 75 mg / ml, more preferably from 40 mg / ml to 60 mg / ml, even more preferably from 45 mg / ml to 55 mg / ml, such as 50 mg / ml.

[0295] As noted above, the nitrogen donor may be any suitable nitrogen donor, which can be used in the Hantzsch pyridine synthesis. Suitable nitrogen donors are well know to the skilled person. Non-limiting examples of a nitrogen donor, which can be used in the process of the present invention, are ammonia and ammonium salts, such as ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate.

[0296] Accordingly, in some embodiments, the nitrogen donor is ammonia.

[0297] In some embodiments, the nitrogen donor is an ammonium salt.

[0298] In some embodiments, the nitrogen donor is an ammonium salt selected from the group consisting of ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate and diammonium sulfate.

[0299] In some embodiments, the nitrogen donor is ammonium acetate. The concentration of the nitrogen donor in the formulation is not particularly limited, and may be adapted as the need requires. Generally, the nitrogen donor, such as an ammonium salt, may be present in a concentration ranging from 0.5 M to 8 M.

[0300] In some embodiments, the nitrogen donor, such as an ammonium salt, is present in a concentration ranging from 0.5 M to 8 M.

[0301] In some embodiments, the nitrogen donor, such as an ammonium salt, is present in a concentration ranging from 1 M to 5 M, preferably from 1 M to 4 M, more preferably from 1.5 M to 2.5 M, such as 2 M.

[0302] The formulation may further comprise additives, such as one or more stabilizer, and one or more crystallization inhibitor, which further improve the functional properties of the formulation.

[0303] Accordingly, in some embodiments, the formulation further comprises a stabilizer, such as a polar aprotic solvent. A non-limiting example of such stabilizer is Dimethyl sulfoxide (DMSO). The concentration of the stabilizer in the formulation is not particularly limited, and may be adapted as the need requires. Generally, the stabilizer may be present in a concentration ranging from 5 % v / v to 25% v / v. In some embodiments, the stabilizer is present in a concentration ranging from 15% v / v to 25% v / v, such as 20% v / v.

[0304] In some embodiments, the formulation further comprises a crystallization inhibitor, such as a water-soluble polymer. A non-limiting examples of such crystallization inhibitor is polyvinylpyrrolidone (PVP), and in particular a PVP selected from the group consisting of PVP K12, PVP K25, PVP K30, PVP K60, and PVP K90. The concentration of the crystallization inhibitor is not particularly limited, and may be adapted as the need requires. Generally, the crystallization inhibitor may be present in a concentration ranging from 0.1% w / v to 1% w / v. In some embodiment, the crystallization inhibitor is present in a concentration ranging from 0.2% w / v to 0.6% w / v, preferably from 0.3% w / v to 0.5% w / v, such as 0.4% w / v.

[0305] The formulation, if an aqueous formulation or a reconstituted lyophilized formation, preferably has a pH ranging from 6.0 to 9.0. The pH can be adjusted to the desired value using any appropriate acidifying or alkalizing agent well known to the skilled person. In some embodiments, the formulation has a pH ranging from 6.5 to 8.5. In some embodiments, the formulation has a pH ranging from 6.5 to 7.5. In some embodiments, the pH is 7.0.

[0306] The formulation of the present invention can be used for detecting formaldehyde in a sample.

[0307] The present invention provides in a further aspect a kit comprising a compound of general formula (I) as defined herein, a nitrogen donor and a reagent comprising an alcohol oxidase or alcohol dehydrogenase, and optionally a catalase. It is understood that all details provided above with respect to the compound of general formula (I), the nitrogen donor and the reagent comprising an alcohol oxidase or alcohol dehydrogenase, and optionally a catalase, especially as disclosed in the context of the formulation of the present invention, apply mutatis mutandis to the kit of the present invention.

[0308] The components of the kit may each be provided in a separate container, or as the specific case may be, may be provided together with one or more of the other components in one container. For example, in some embodiments, the compound of general formula (I) and the nitrogen donor are provided together in a formulation, such as a formulation as detailed above, contained in one container, while the reagent comprising an alcohol oxidase or alcohol dehydrogenase, and optionally a catalase, is provided in another container. This setting, however, also encompasses a setting in which the catalase is provided separately from the reagent comprising an alcohol oxidase or alcohol dehydrogenase in a yet further container. If a catalase is present, the catalase is preferably provided in the same container as the alcohol oxidase or alcohol dehydrogenase, since it provides a stabilizing effect on the latter enzymes.

[0309] The reagent comprising an alcohol oxidase or alcohol dehydrogenase may take any suitable form. The reagent may be a liquid, preferably aqueous, solution. It may also be a powder or lyophilized solution. Preferably, the reagent is an aqueous solution. An aqueous solution may be prepared by adding the enzyme(s) in their dried (powdered) form to a suitable aqueous solvent, such as water. The aqueous solution may also be prepared by reconstituting a lyophilized solution containing the enzyme(s) in, e.g. water or any other suitable aqueous solvent.

[0310] The reagent, if an aqueous solution or a reconstituted lyophilized solution, preferably has a pH ranging from 6.0 to 9.0. The pH can be adjusted to the desired value using any appropriate acidifying or alkalizing agent well known to the skilled person. In some embodiments, the reagent has a pH ranging from 6.5 to 8.5. In some embodiments, the reagent has a pH ranging from 7.0 to 8.0. In some embodiments, the pH is 7.5.

[0311] The concentration of the alcohol oxidase or alcohol dehydrogenase is not particularly limited, and may be adapted as the need requires. Generally, the alcohol oxidase or alcohol dehydrogenase is present in a concentration ranging from 0.5 ll / rnl to 20 U / rnl. In some embodiments, the alcohol oxidase or alcohol dehydrogenase is present in a concentration ranging from 1 ll / rnl to 10 ll / rnl, preferably from 2 ll / rnl to 8 ll / rnl, more preferably from 3 ll / rnl to 6 ll / rnl, such as 4 ll / rnl or 5 ll / rnl.

[0312] The concentration of the catalase is not particularly limited, and may be adapted as the need requires. Generally, the catalase is present in a concentration ranging from 0.05 kll / ml to 5 kll / ml. In some embodiments, the catalase is present in a concentration ranging from 0.5 kll / ml to 2.5 kU / ml, preferably from 0.5 kU / ml to 1.5 kU / ml, such as 1 kU / ml. The kit of the present invention can be used for detecting methanol in a sample.

[0313] It will be appreciated that the above concentrations are given with respect to the formulation(s) and reagent(s) being in liquid, preferably aqueous, form. However, these concentrations are not limited to this state. They also encompass the concentrations of the various components in their dry or concentrated state, such as in mixtures of solids, dried residues, or lyophilized forms. Naturally, in the dry state, the concentrations are higher due to the absence of solvent or reduction of volatile components. Upon preparation of the liquid formulation or reagent using an appropriate aqueous solvent, these dry state concentrations will result in the defined liquid concentrations.

[0314] Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

[0315] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

[0316] Brief description of the drawings

[0317] Legend to Figure 1 : Comparison of different p-ketocarbonyl compounds in a formaldehyde- detection reaction. A reagent (SR) containing a specified p-ketocarbonyl compound and ammonium acetate with a specified pH was added to a buffered solution (CB), after which an aqueous formaldehyde solution (sample) was added. The diyhdropyridines formed by the Hantzsch reaction (Hantzsch dihydropyridine synthesis) were determined spectrophotometrically at different wavelengths. Duplicate measurements were made on a microplate reader. Final concentrations of formaldehyde in the reaction mixtures were 0 mg / mL, 15 mg / mL, 30 mg / mL, 45 mg / mL, and 60 mg / mL, respectively. The time scale depicted in the Figure starts with sample addition (t = 0 s).

[0318] Legend to Figure 2: Comparison of different p-ketocarbonyl compounds in a methanol detection assay. Formaldehyde is generated enzymatically by methanol oxidation using alcohol oxidase, and formaldehyde detection is effected by Hantzsch reaction (Hantzsch dihydropyridine synthesis) with p-ketocarbonyl compound and ammonium acetate. Enzymatic oxidation and Hantzsch reaction were running in parallel. A reagent ( SR[i] or SR[ii] ) containing P-ketocarbonyl compounds and ammonium acetate with the specified pH was added to a buffered solution containing alcohol oxidase (R1) after which an aqueous methanol solution (sample) was added. The diyhdropyridines formed by the Hantzsch reaction were measured spectrophotometrically at different wavelengths. Duplicate measurements at the Cedex Bio HT analyzer. Methanol concentrations are related to the added sample (150 pL R1 + 40 pL SR, 2 pL sample + 10 pL water). The time scale depicted in the Figure starts with sample addition (t = 0 s).

[0319] Legend to Figure 3: Background absorbance increase (AA1) at 378 nm of different HR formulations (0a, Ob, 1 , 2, 4-1 , 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 5, 6, 7, 9, 10, 11 , 12) in a methanol-detection assay over accelerated storage at 25 °C at different time points. For details, see Example 3. The signals correspond to absorbing by-products formed from the ammonium salt and the N-Acetoacetylanthranilic acid during storage. AA1 is not relevant for the methanol quantification (formaldehyde detection via the Hantzsch dihydropyridine synthesis) but indicative for the ageing of the HR solutions.

[0320] Legend to Figure 4: . Methanol signal (AA2) with different HR formulations (0a, Ob, 1 , 2, 4-1 , 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 5, 6, 7, 9, 10, 11 , 12) in a methanol-detection assay over accelerated storage at 35 °C at different time points for two methanol concentrations 0 g / L and 5 g / L in the sample. For details, see Example 3. The signal corresponds to the dihydropyridine formed by the Hantzsch reaction (Hantzsch dihydropyridine synthesis) between formaldehyde, ammonia and N-(acetoacetyl)anthranilic acid and can be related to a methanol concentration when calibrated. The progressive loss of the signal is a result of the depletion of available reaction equivalents due to accelerated ageing of the HR formulations.

[0321] Examples

[0322] Example 1 : Screening of p-carbonyl compounds capable of reacting in a Hantzsch dihydropyridine synthesis with formaldehyde

[0323] In summary, data are provided indicating that N-(acetoacetyl)anthranilic acid (N-AAAA) has superior properties in a sensitive reaction to detect formaldehyde.

[0324] Each Hantzsch dihydropyridine synthesis reagent was prepared with a p-ketocarbonyl compound selected from the group consisting of acetoacetamide (AA), N- (acetoacetyl)anthranilic acid (N-AAAA), potassium N-(acetoacetyl)sulfanilate (K-AAsA), and acetylacetone (AA).

[0325] For each p-ketocarbonyl compound, two screening reagents (SR) designated SR[i] and SR[ii] were prepared. The respective p-ketocarbonyl compound was at a concentration of 226 mM in a buffer containing 4 M ammonium acetate, with the respective SR[i] having pH 6.8 and the SR[ii] having pH 7.8, adjusted with NaOH or with glacial acetic acid.

[0326] For the screening a dilution series of formaldehyde in H2O was provided comprising the concentrations 0.25 g / L, 0.5 g / L. and 1 g / L.

[0327] Screening of the p-ketocarbonyl compounds was performed in parallel (a) in water and (b) in a more complex buffer designated CB. The latter was composed of 100 mM Potassium Phosphate (K-PO4), 200 mM Potassium Chloride (KCI), 500 mM Trehalose, 0.2% (w / v) bovine serum albumin, and 0.09% (w / w) Oxy-Pyrion, adjusted to pH 7.5.

[0328] The screening of the p-ketocarbonyl compounds was performed by setting up detection reactions in a microwell format using 96-well microwell plates (Corning 96-well Clear Flat Bottom UV-Transparent Microplate, Cat. 3635). For each reaction a mix was prepared in a microwell, the mix containing 150 pL water or CB, and 40pL of the respective SR[i] or SR[ii]; the reaction was started by adding (and mixing) 12 pL of a formaldehyde sample from the dilution series. Controls were included with 12pL of water added, instead of a formaldehyde sample. In exemplary final mixes the concentration of the respective p-ketocarbonyl compound was 45mM. Other concentrations are suitable as well.

[0329] Photometric measurements were performed to trace formation of Hantzsch reaction products; absorbance was recorded over the course of 30 min with a microplate absorbance spectrophotometer (BioTek / Agilent Synergy HT microplate reader). Measurements were taken at 378 nm and 405 nm, while a temperature of 25 °C was maintained throughout the reaction time. The recordings depicted in Figure 1 reflect the progression of dye product formation as a result of the Hantzsch reaction.

[0330] Substantially similar results were obtained with water instead of CB.

[0331] Example 2: Screening of the p-carbonyls used in the Hantzsch Di hydropyridine synthesis for formaldehyde detection from methanol show that N- Acetoacetylanthranilic acid is superior

[0332] Each Hantzsch dihydropyridine synthesis reagent was prepared with a p-ketocarbonyl compound selected from the group consisting of acetoacetamide (AA), N- (acetoacetyl)anthranilic acid (N-AAAA), potassium N-(acetoacetyl)sulfanilate (K-AAsA), and acetylacetone (AA).

[0333] For each p-ketocarbonyl compound, two screening reagents (SR) designated SR[i] and SR[ii] were prepared. The respective p-ketocarbonyl compound was at a concentration of 226 mM in a buffer containing 4 M ammonium acetate, with the respective SR[i] having pH 6.8 and the SR[ii] having pH 7.8, adjusted with NaOH or with glacial acetic acid. The measurements were conducted using the Cedex Bio HT analyzer (Roche Diagnostics) at two wavelengths, 378 nm and 409 nm, at a controlled temperature of 35-37°C. The pipetting protocol for each reaction in the experiment was as follows: 150 pL of Reagent 1 (R1) was combined with 40 pL of Reagent SR ( SR[i] or SR[ii] ) in each cuvette.

[0334] Reagent 1 had the following composition: 4 U / rnL Alcohol Oxidase from Pichia pastoris / Komagataella phaffii (AOX), 1 kll / mL Catalase, 100 mM Potassium Phosphate (K- PO4), 200 mM Potassium Chloride (KCI), 500 mM Trehalose, 0.2% (w / v) bovine serum albumin, and 0.09% (w / w) Oxy-Pyrion, adjusted to pH 7.5. Reagent SR consisted of 4 M Ammonium Acetate (NH4OAC) buffer and 226 mM of a p-ketocarbonyl compound (226 mM corresponds to 50 mg / mL of N-AAAA). The pH was adjusted to either 6.8 or 7.8 ( SR[i] and SR(ii) respectively) with NaOH or with glacial acetic acid.

[0335] The mixture was allowed to equilibrate to the reaction temperature and for approx. 2.5 min after which 2 pL of the sample and 10 pL of water were added to the cuvette and absorbance was recorded for 9 min. The samples consisted of aqueous methanol solutions (0, 3.33 or 5 g / L).

[0336] The results are depicted in Figure 2.

[0337] Example 3: Formulation optimization of the Hantzsch dihydropyridine reagent for formaldehyde / methanol detection

[0338] With the goal to compare different formulations of the Hantzsch reagent (Hantzsch dihydropyridine synthesis) for formaldehyde / methanol detection, Reagent 1 was kept unchanged, but the composition of the Hantzsch reagent [HR] was varied.

[0339] Reagent 1 (R1) contained 4 - 5 U / rnL of alcohol oxidase, 1 kll / mL of catalase, 100 mM of K- PO4 buffer, 150 mM of KCI, 500 mM trehalose, 0.2 % of BSA, 0.9 % of Oxy-pyrion with a pH of 7.5.

[0340] Individual formulations of Hantzsch reagent (HR) were composed as follows:

[0341] 0a: 50 mg / mL N-AAAA, 4 M ammonium acetate, pH 6.8;

[0342] 0b: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, pH 6.8;

[0343] 1 : 50 mg / mL N-AAAA, 4 M ammonium acetate, 10 % DMSO, pH 6.8;

[0344] 2: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, 10 % DMSO, pH 6.8;

[0345] 3: 50 mg / mL N-AAAA, 4 M ammonium acetate, 0.1 % Sodium deoxycholate, pH 6.8;

[0346] 4: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, 0.1 % Sodium deoxycholate, pH

[0347] 6.8;

[0348] 4-1 : 50 mg / mL N-AAAA, 4 M ammonium acetate, 0.4 % PVP K90F (PubChem SID

[0349] 481110346), 10 % DMSO, pH 7.0; 4-2: 50 mg / mL N-AAAA, 4 M ammonium acetate, 0.4 % PVP K90F, 20 % DMSO, pH 7.0;

[0350] 4-3: 50 mg / mL N-AAAA, 4 M ammonium acetate, 20 % DMSO, pH 7.0;

[0351] 4-4: 50 mg / mL N-AAAA, 2 M ammonium acetate, 0.4 % PVP K90F, 10 % DMSO, pH 7.0;

[0352] 4-5: 50 mg / mL N-AAAA, 2 M ammonium acetate, 0.4 % PVP K90F, 20 % DMSO, pH 7.0;

[0353] 4-6: 50 mg / mL N-AAAA, 2 M ammonium acetate, 20 % DMSO, pH 7.0;

[0354] 4-7: 50 mg / mL N-AAAA, 4 M ammonium acetate, 0.4 % PVP K90F, 10 % DMSO, pH 6.8;

[0355] 4-8: 50 mg / mL N-AAAA, 4 M ammonium acetate, 0.4 % PVP K90F, 20 % DMSO, pH 6.8;

[0356] 5: 50 mg / mL N-AAAA, 4 M ammonium acetate, 50 mM Na-Citrate, pH 6.8;

[0357] 6: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, 50 mM Na-Citrate, pH 6.8;

[0358] 7: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, 0.1 % PVP10 (Polyvinyl pyrrolidone, with average mol. weight 10,000), pH 6.8;

[0359] 8: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, 0.1 % PVP10, pH 6.8 (not pursued further, PVP10 has lowered solubility here);

[0360] 9: 50 mg / mL N-AAAA, 4 M ammonium acetate, 20 % DMSO, pH 6.8;

[0361] 10: 50 mg / mL N-AAAA, 4 M ammonium acetate, pH 7.0;

[0362] 11 : 50 mg / mL N-AAAA, 2 M ammonium acetate, pH 6.8;

[0363] 12: 50 mg / mL N-AAAA, 4 M ammonium phosphate buffer, pH 7.5

[0364] The different HRs were filled into Cedex Bio HT cassette segment bottles and incubated in a temperature-controlled incubator at 35 °C. For the periodic measurements, the bottles were taken out, assembled with fresh R1 segment bottles to cassettes and subjected to the measurement at the Cedex® Bio HT. After each measurement series the cassettes were disassembled and the HR segment bottles were transferred to the incubator again. Measurement series and incubation was done for up to 71 d and a measurement series usually stopped prematurely when crystal formation (see below) occurred.

[0365] The HRs were also filled into transparent 5 mL reaction tubes made out of polypropylene (Eppendorf AG), incubated in a temperature-controlled incubator at 35 °C and checked day by day for the appearance of precipitates or crystals in the solutions or at the tube wall.

[0366] The Hantzsch reagents (HR) were analyzed spectrophotometrically using a sequential pipetting setting on the Cedex® Bio HT (Roche Diagnostics) at a controlled temperature of 37 °C with separated alcohol oxidase reagent (R1) and Hantzsch reagents (HR). The device was programmed so that 150 pL of reagent 1 (R1) and 2 pL of a methanol sample solution (5 g / L or 0 g / L) and 10 pL of water are sequentially added to a microcuvette and equilibrated for ~3 min (170 s). 40 pL of one particular HR is then added and the absorbance is recorded for ~ 6 min (371 s) at 378 nm and 37 °C.

[0367] For the analysis, the absorbance difference 1 (AAi) reflects the background absorbance of the HR and is formed by the volume-corrected difference of the absorbance after SR addition (see text above) and the absorbance after R1 addition to the cuvette. The absorbance difference 2 (AA2) reflects the signal generated by the dihydropyridine synthesis of the HR and is formed by the volume-corrected difference of the absorbance after the complete reaction mix had incubated for 371 s and the absorbance after HR addition to the cuvette (see text above). AA1 and AA1 over different time points were fitted using a 2P-linear model (JMP, JMP Statistical

[0368] Discovery LLC) to obtain slopes for comparison. Note that for most of the HR formulations, the incubation in both the segment bottles and reaction tubes was prematurely stopped when crystals or precipitate was observed in the reaction tube.

Claims

Claims1. Use of a compound of general formula (I) or a salt thereof in a Hantzsch reactionwhereinR1is -H or -Me; at least one of R2, R3, R4, R5and R6is a carboxylic acid substituent ( COOH), preferably is one carboxylic acid in the R2or R6position; and any one of R2, R3, R4, R5and R6, if not equalling to COOH, is each independently selected from the group consisting of - H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7(CH2)i. 6COOH, -CONR7(CH2)I.6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i.6SO3H; andR7is, if applicable, -H or -Me, preferably is -H.

2. Use according to claim 1 , wherein the compound of general formula (I) is a compound of formula (la)whereinR1is -H or -Me;R2, R3, R4, and R5are each independently selected from the group consisting of -H, -R7is, if applicable, -H or -Me, preferably is -H; or wherein the compound of general formula (I) is a compound of formula (lb)wherein R1is -H or -Me; or wherein the compound of general formula (I) is a compound of formula (Ic)wherein R1is -H or -Me.

3. Compound of general formula (II)whereinR1’ is -H or -Me; at least one of R2’, R3’, R4’, R5’ and R6’ is a carboxylic acid substituent (COOH), preferably is one carboxylic acid in the R2’ or R6’ position; and any one of R2’, R3’, R4’, R5’ and R6’, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, - CONR7(CH2)I.6COOH, -CONR7’(CH2)I-6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i- 6SO3H;R7’ is, if applicable, -H or -Me, preferably is -H;R1” is -H or -Me;at least one of R2”, R3”, R4”, R5” and R6” is a carboxylic acid substituent (COOH), preferably is one carboxylic acid in the R2” or R6” position; and any one of R2”, R3”, R4”, R5” and R6”, if not equalling to COOH, is each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, - CONR7(CH2)I.6COOH, -CONR7”(CH2)I-6SO3H, -SO3H, -Cl, -F, -CF3, and - NMe2+(CH2)i-6SO3H;R7” is, if applicable, -H or -Me, preferably is -H.

4. Compound according to claim 3, wherein the compound is a compound of formula (Ila)whereinR1’ is -H or -Me;R2’, R3’, R4’, and R5’ are each independently selected from the group consisting of -H, - OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, -CONR7’(CH2)I-6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i-6SO3H;R7’ is, if applicable, -H or -Me, preferably is -H;R1” is -H or -Me;R2”, R3”, R4”, and R5” are each independently selected from the group consisting of -H, -OH, -Me, -OMe, -OEt, -NMe2, -NMe3+, -NEt2, -NEt3+, -CONH2, -CONR7’(CH2)I.6COOH, -CONR7’(CH2)I-6SO3H, -SO3H, -Cl, -F, -CF3, and -NMe2+(CH2)i-6SO3H; andR7” is, if applicable, -H or -Me, preferably is -H; or wherein the compound is a compound of formula (lib)wherein each R1is independently -H or -Me; or wherein the compound is a compound of formula (He)wherein each R1is independently -H or -Me.

5. Use of a compound according to claim 3 or 4 as a dye, particularly wherein the compound is used as a dye in the detection of formaldehyde or a related compound or particularly wherein the compound is used as a dye in the detection of methanol.

6. Process for the production of a compound of general formula (II) according to claim 3 or 4, the method comprising: a) reacting a compound of general formula (I) as defined in claim 1 or 2 with formaldehyde in the presence of a nitrogen donor in a suitable solvent to allow formation of the compound of general formula (II); and b) optionally isolating the compound of formula (II) from the reaction.

7. Process according to claim 6, a) wherein the compound of general formula (II) is a compound of formula (He), and the compound of general formula (I) is a corresponding compound of formula (Ic); b) wherein the compound of general formula (II) is a compound is defined in that R1is -H;c) wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof, particularly wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate; d) wherein the solvent is water; e) wherein the reaction is carried out at a temperature ranging from 15 °C to65 °C, such as from 20 °C to 40 °C, particularly at a temperature ranging from 30 °C to 40 °C, especially at 37 °C; and / or f) wherein the reaction is carried out for a period of time ranging from 10 seconds to 60 minutes, particularly for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes, especially for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

8. Method for the detection of formaldehyde in a sample, the method comprising: a) contacting a sample with a compound of general formula (I) as defined in claims 1 or 2 and a nitrogen donor to form a reaction mixture and incubating the reaction mixture; and b) determining whether a compound of formula (II) as defined in claims 3 or 4 is or has been formed during the course of incubating the reaction mixture, wherein the formation of the compound of formula (II) is indicative of the presence of formaldehyde in the sample.

9. Method according to claim 8, wherein a) wherein the compound of general formula (II) is a compound of formula (He), and the compound of general formula (I) is a corresponding compound of formula (Ic); b) wherein the compound of general formula (II) is a compound is defined in that R1is -H; c) wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof, particularly wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate;d) wherein the reaction is carried out at a temperature ranging from 15 °C to65 °C, such as from 20 °C to 40 °C, particularly at a temperature ranging from 30 °C to 40 °C, especially at 37 °C; and / or e) wherein the reaction is carried out for a period of time ranging from 10 seconds to 60 minutes, particularly for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes, especially for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

10. Method for the detection of methanol in a sample, the method comprising: a) contacting a sample with an alcohol oxidase or alcohol dehydrogenase to form a first reaction mixture and incubating the first reaction mixture; b) optionally, subjecting the incubated first reaction mixture to peroxide scavenging by adding a catalase; c) contacting the incubated reaction mixture obtained in step a) or b) with a compound of general formula (I) as defined in claim 1 or 2 and a nitrogen donor to form a second reaction mixture and incubating the second reaction mixture; and d) determining whether a compound of formula (II) as defined in claim 3 or 4 is or has been formed during the course of incubating the second reaction mixture, wherein the formation of the compound of formula (II) is indicative of the presence of methanol in the sample.

11. Method according to claim 10, wherein a) wherein the compound of general formula (II) is a compound of formula (He), and the compound of general formula (I) is a corresponding compound of formula (Ic); b) wherein the compound of general formula (II) is a compound is defined in that R1is -H; c) wherein the nitrogen donor is ammonia, an ammonium salt, or a combination thereof, particularly wherein the ammonium salt is ammonium acetate, ammonium phosphate, ammonium dihydrogen phosphate, diammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, ammonium hydrogen sulfate or diammonium sulfate;d) wherein the first reaction mixture is incubated at a temperature ranging from15 °C to 65 °C, such as from 20 °C to 40 °C, particularly at a temperature ranging from 30 °C to 40 °C, especially at 37 °C; e) wherein the first reaction mixture is incubated for a period of time ranging from 10 seconds to 60 minutes, particularly for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes, especially for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes; f) peroxide scavenging is carried out at a temperature ranging from 15 °C to65 °C, such as from 20 °C to 40 °C, particularly at a temperature ranging from 30 °C to 40 °C, especially at 37 °C; g) wherein the peroxide scavenging is carried out for a period of time ranging from 10 seconds to 60 minutes, particularly for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes, especially for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes; h) wherein the second reaction mixture is incubated at a temperature ranging from 15 °C to 65 °C, such as from 20 °C to 40 °C, particularly at a temperature ranging from 30 °C to 40 °C, especially at 37 °C; and / or i) wherein the second reaction mixture is incubated for a period of time ranging from 10 seconds to 60 minutes, particularly for a period of time ranging from 1 minute to 60 minutes, such as from 10 minutes to 30 minutes, especially for a period of time ranging from 10 minutes to 60 minutes, such as from 20 minutes to 40 minutes.

12. The method according to any one of claims 8-11 , a) wherein the step of determining of whether a compound of formula (II) is or has been formed comprises measuring the absorbance using a spectrophotometer, such as a UV-vis spectrophotometer, or an analyzer system comprising a spectrophotometric unit, particularly wherein the absorbance is measured at a wavelength in the range from 350 nm to 420 nm, preferably in the range from 365 nm to 390 nm, more preferably at 378 nm; b) wherein the step of determining of whether a compound of formula (II) is or has been formed comprises measuring visible spectrum fluorescence using a fluorometer or an analyzer system comprising a fluorometric unit, particularly wherein the fluorescence is measured using a excitation wavelength in the range from 350 nm to 420 nm, preferably in the range from 365 nm to 390 nm, more preferably at 378 nm, and an emission / detection wavelength in the rangefrom 420 nm to 550 nm, preferably in the range from 450 nm to 480 nm, more preferably at 470; and / or c) wherein the compound of general formula (I) and nitrogen donor are provided together in the form of an aqueous formulation.

13. The method according to any one of claims 8-11 , wherein the compound of general formula (I) and nitrogen donor are provided together in the form of an aqueous formulation and wherein the formulation further comprises a) a stabilizer, particularly wherein the stabilizer is a polar aprotic solvent, preferably Dimethyl sulfoxide (DMSO); and b) optionally a crystallization inhibitor, particularly wherein the crystallization inhibitor is a water-soluble polymer, preferably polyvinylpyrrolidone (PVP), more preferably PVP K90, especially wherein the aqueous formulation has a pH ranging from 6.0 to 8.0, preferably from 6.5 to 7.5, such as about 7.0.

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