Active Derivatization Of Mono And Dicarboxylic Acids Biology Essay

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Many methods for the determination of short and medium chain dicarboxylic acids were developed [1, 2, 4]. A method has been selected through literature survey for fluorescence active derivatization of carboxylic acids (Oxalic Acid, Adipic acid, Malonic acid, Succinic acid, Glutaric acid, adipic acid, Pimelic acid, Suberic acid, Azelaic acid, Cis-pinonic acid, Pinic acid, Syringic acid, Pthalic acid ) in order to achieve high sensitivity on spectroflurometer . Good separation of these corresponding derivatized acids (C2-C10) is possible with reverse phase liquid chromatography by isocratic elution when an ODS column is used in reverse phase Liquid chromatography [1].

Emission spectra of a flurophore mainly depends on its chemical structure and the solvent in which flurophore exists [7]. An emission spectrum was generally a representative of fluorescence spectral data [7]. "A fluorescence emission spectrum was a plot of the fluorescence intensity versus wavelength (nanometers) or wave number (cm-1)'' [7].

In a derivatization reaction, when 4-(1-pyrene)butyric acid hydrazide (PBH) reagent in DMSO (Dimethyl sulfoxid) is used according to an appropriate method[1, 2], along with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) with pyridine in DMSO solvent, the fluorescence active pyrene derivatives of corresponding carboxylic acid are produced. This reaction was reported by Notha et al. and co-workers [1, 2, 3].

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Pyrene labeled compounds provides fluorescence emission spectra. Pyrene molecule is the basic source of fluorescence in "PBH derivatives" (in further discussions pyrene in PBH derivative refers to "pyrene labeled compounds"). When dicarboxylic acids are derivatized the corresponding ester can produce an ''excimer''. The derivatized dicarboxylic acid to 4-1(pyrene) butyric acid N-hydroxysucciniimide ester (Fig 1) provides an intra molecular fluorescence emission in the range of 450-520 nm [1, 4]. An acid containing mono carboxylic (functional) group can be derivatized to mono pyrene labeled ester and provide a monomer fluorescence spectra (370-420 nm) [1]. When dicarboxylic acids are derivatized to mono pyrene labeled esters, the derivatives are also expected to show fluorescence emission in monomer region of spectra [3].

"Excimer" is a complex between excited molecule and its corresponding ground energy state due to the interaction between these two energy states [3]. ''Pyrene'' fluorescence properties were discovered by Forster and Kasper [3].

Monomers and excimer of pyrene derivatives provide some advantages comparative to other fluorescence active flurophores. Fluorescence bands, between monomer and excimer are usually easy to recognize. These fluorescence bands were well alienated from each other and provide high quantum yield (0.1-0.9). Life time of monomer and excimer fluorescence is longer which makes possible for a spectroflurometer to quantify fluorescence signals more effectively. Pyrene as a fluorescent source has another advantage that usually a simple reaction is involved for its insertion to a molecule [3].

Intra molecular excimer fluorescence has enough selectivity between mono and dimers (dicarboxylic derivatives) [1, 2, 4]. Yamaguchi et al provides a scheme to discriminate between mono and multi derivatives of pyrene chromophors in the fluorescence spectrum. According to ''Yamaguchi's scheme'' mono pyrene derivative and reagent itself provide fluorescence emission at 375 nm while multi pyrene derivative provide emission at 475 nm, when all of these derivatives are excited at the same wavelength (345 nm) [4]. There are several unwanted compounds expected during this chromatographic study, these compounds can be separated through a good liquid chromatographic column and with selective fluorescence emission spectra [1].

Inorder to produce an "excimer", a molecule require minimum two pyrene chromophores. The two chromophores of pyrene, when attached to a molecule should have some conformational changes to produce pi-symmetry in order to display fluorescence in excimer region [3]. Excimer can also be formed when two or more molecules, provide pyrene chromophores the same symmetry as described in previous paragraph

Pyrene chromophores bear some disadvantages which limit its fluorescence yield; because pyrene molecule has a long hydrophobic chain its order can be changed in water. Pyrene is very sensitive to some quentures i.e. molecular Oxygen can act as a quenture because of longer fluorescence decay of pyrene it is easy for a quenture to capture and reduce fluorescence yield [3].

This work was selected to perform with the same analytes (C3-C10) those were successfully derivatized with BSTFA, except Oxalic acid (C2). Samples after extraction were in aqueous media due to extraction from three phase hf-lpme technique. BSTFA is water sensitive, so there were some problems in repeatability of results to evaporate water till dryness. This project was aimed to provide water insensitive derivatization which is much suitable with three phase HF-LPME[11].

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A reagent dimer was produced in the derivatizing reaction (Fig. 1c) as a byproduct and this dimer produce intense excimer fluoresces [2] which can interfere with the detection of dicarboxylic acids. A method to decrease [2] interference of reagent dimer in the chromatographic analysis, by the dilution of the derivatized solution with aqueous THF, was also applied.

Current derivatization reaction was reported with different concentration of EDC and Pyridine to minimize generation of side products and to enhance efficiency [2].

a)b)

C)

Fig. 1.1 [2] - Derivatization of Dicarboxylic acids with PBH, Scheme (a) Drivatization reaction of Dicarbxylic acid with PBH; (b) degradation of PBH to 4-(1-pyrene)butanoic acid; (c) reaction of PBH and 4-(1-pyrene)butanoic acid to produce a dimer.

EDC is a water soluble compound and its reaction can take place without an organic solvent. EDC is very effective to generate a new amide bond due to the reaction between carboxylic acids and primary amines. The mechanism for this reaction was not clear i.e. EDC reacts with carboxylic acid to form an unstable intermediate O-acylisourea (Fig 1.2). This intermediate was very active and reacts instantly with primary amine (neucleophile) to form an amide bond [8].

Figure 1.2 [8]- Reaction of EDC with carboxylic acids to create an active-ester intermediate, primary amine act as a nucleophile, Isourea by-product was produced along with a new amide bond

Time of flight (TOF) Mass spectrometer was used for the identification of compounds that were proposed to produce through the (Fig 1) derivatization procedure (discussed already). TOF mass spectrometer, having orthogonal accelerator provides excellent efficiency [5]. Full scan acquisitions were possible with TOF mass spectrometer. TOF mass spectrometer has provided high sensitivity in scan mode because the detection of entire mass was achieved in the same time [6]. TOF mass spectrometer has high efficiency (2-5 ppm) and high power for mass resolution [5]. All previously described qualities make TOF a very effective tool for confirmation of trace amount of Organic compounds [5].

2- Method:

2.1-Reagents:

PBH (pure) and 97% EDC (HCL) were purchased from Sigma Aldrich. DMSO was purchased from Fischer Scientific, Pyridine was purchased from Aldrich. Acetonitrile, Methanol and Tetrahydrofurane (THF) were of gradient grade and purchased from 'Honey well'. Milli Q water was obtained from Millipore water system. Description of individual analytes, purity, molecular formulas and pKa values were given in table 2.1.

Table 2.1- Analytes description, Source and purity (%):

Sr. No.

Chemicalname

Molec. formula

Purchased

from

pKa Values

Purity (%)

1

Oxalic Acid

C2H2O4

Sigma Aldrich

1.23, 4.19(36)

99

2

Malonic Acid

C3H4O4

Aldrich

2.83, 5.69 (36)

99

3

Succinic Acid

C5H6O4

Fluka

4.19, 5.48 (36)

99.9

4

Glutaric Acid

C5H8O4

Aldrich

4.34, 5.42 (36)

99

5

Adipic Acid

C6H10O4

Fluka

4.34,5.44 (36)

99.5

6

Pimelic Acid

C7H12O4

Aldrich

4.48, 5.42 (36)

98

7

Suberic Acid

C8H14O4

Aldrich

4.52, 5.40 (36)

98

8

Azelic Acid

C9H16O4

Aldrich

4.55, 5.41 (36)

98

9

Cis-Pinonic Acid

C10H16O3

Sigma Aldrich

-

98

10

Pinic Acid

C9H14O4

Sigma Aldrich

-

99

11

Syringic Acid

C9H10O5

Sigma

-

-

12

Pthalic Acid

C8H6O4

Sigma Aldrich

2.98,528(41)

99.5

13

4-Hydroxy benzoic Acid

C7H6O3

Aldrich

4.52,9.23 (35)

99

2.1.1- Solutions:

Stock solutions (100 µg/ml) of Organic acids (C2-C10) were prepared in DMSO. This solution mostly was prepared fresh or otherwise stored at 0 °C and was used within a week. Further dilutions for these analytes (0.1-10 µg/ml) and multi-component mixture (8.3 µg/ml) were prepared in DMSO. Multi component mixture contained all target analytes (1-13 in table 2.1).

2.2- Operational conditions:

2.2.1- Liquid Chromatographic detection:

A gradient LC system (Agilent 1100 series; Germany) was used for chromatographic analysis. Gradient LC system was equipped with Chromatographic pump, an Injector having a '20 µl' sample loop. A reverse phase C18 column (ODS-2-HYPERSIL, 100 x 2.1 mm ID), particle size 5 µm (Thermo Scientific) was used for the separation of target compounds.

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A spectroflourometer (1200 series fluorescence detector; Germany) was fitted with an '8 µl' flow cell. Acetonitrile and water were used as mobile phase in a volumetric ratio 64:36 respectively. The composition of mobile phase was varied as well during different experiments but latter on it was fixed at an optimum level (64:34). Fixed excitation wavelength (345 nm) and emission wavelengths (480 nm) were used for the detection of dicarboxylic acids. For the detection of mono pyrene labeled compounds excitation wavelength was unchanged while emission wavelength was varied in the range of 360-390 nm. Chromatographic results are presented in next section with different emission wavelengths.

Table 2.2- Exact masses (at 100% abundance) of analytes and respective PBH derivatives

Sr No

Analyte

Exact mass of Analytes

Mass of mono pyrene derivative (+1)

Mass of di-Pyrene derivative (+1)

1

Oxalic Acid

90.03

375.06

659.09

2

Malonic Acid

104.01

389.04

673.07

3

Succinic Acid

118.03

403.06

687.09

4

Glutaric Acid

132.04

417.07

701.1

5

Adipic Acid

146.06

431.09

715.12

6

Pimelic Acid

160.07

445.1

729.13

7

Suberic Acid

174.09

459.12

743.15

8

Azelic Acid

188.04

473.07

757.1

9

Cis-Pinonic Acid

184.11

469.14

753.17

10

Pinic Acid

186.09

471.12

755.15

11

Syringic Acid

198.05

483.08

767.11

12

Pthalic Acid

166.03

451.06

735.09

13

4-Hydroxy benzoic Acid

138.03

423.06

707.09

2.2.2- TOF mass spectrometer:

TOF mass spectrometer was interfaced with a gradient liquid chromatographic system. TOF mass spectrometer (Pe Sciex, API QSTAR -PULSAR) was used to confirm pyrene derivatives. TOF mass spectrometer was operated in positive ionization mode in the range of 400 -900 m/z Gradient LC System (Ultimate) was used having '20 µl' sample loop and fitted with an auto sampler (Famos). Methanol and Mili Q water were used as mobile phase solvents. Methanol water ratio (%) at the start were 60:40, after 1.5 min 95:5, after 12.5 min till the end of chromatographic run (20 min) 60:40. Table 2.2 shows exact mass of target analytes (C2-C10) and their respective pyrene labeled (PBH derivatives) iimmide esters [4]. According to the scheme (Fig. 2.1) of derivatization, a mono pyrene derivative was produced by the elimination of single water molecule and two water molecules were eliminated when diimide ester was produced and corresponding mass of molecular ion was given in table 2.2.

2.3- Derivatizing Procedure:

Following dervatization (2.3.1-23.6) procedure were applied in order to obtain better fluorescence signals. EDC solution was prepared in water and other solutions containing analytes (C2-C10), pyridine and PBH were prepared in DMSO.

A pear shaped flask (glass vial) having a '100 µl' capacity was used as a reaction vial. Standard solution (100 µg/ml) in 20 µl amount was added in the flask, 10 µl of 20% (v/v) pyridine solution was successively added along with 10 µl of 2M EDC. 20 µl of 5mM PBH solution was added finally and then reaction vial was tightly closed and was put in oven at 40 °C for 60 min. After cooling this mixture, at less than 0 °C for 15min, sample was injected directly to the HPLC system. In the similar way solution containing individual analytes (100 µg/ml) and a multi component mixture (table 2.1) having strength 8.33 µg/ml were derivatized and injected in the similar method.

An amber colored flask with 1.5 ml capacity was used as a reaction vial. 25 µl of 20 % (v/v) pyridine solution was added successively along with 25 µl of 2M EDC. 50 µl of 5mM PBH solution was added and than reaction vial was tightly closed and was put in oven at 40 °C for an Hour. This solution was cooled (-5 - 0 0C) for 15 min and injected directly to the HPLC system.

Same procedure as given in section 2.3.2 except 40% pyridine was used.

Same procedure as given in section 2.3.2 except 60 °C temperature was used for derivatizaion for an hour.

Same procedure as given in section 2.3.2, except after 15 min cooling to -5 - 0 0C this sample was further diluted 100 times and 1000 times with 50% aqueous THF prior injecting to LC system.

In 1.5 ml capacity flask (2.3.2), 25 µl of 40 % (v/v) pyridine solution was added along with 25 µl of 1M EDC. 50 µl of 50 mM PBH solution was added and than reaction vial was tightly closed and was put in oven at 80 °C for an Hour. After 15 min cooling to -5 - 0 oC temperature, this sample was further diluted 10 times and 1000 times with aqueous 50% THF.

3- Results

3.1- Detection of mono and di-pyrene labeled compound in fluorescence emission Spectra:

Fig. 3.1- 3.2 show emission spectra from monomer fluorescence region of successfully derivatized mono pyrene labeled acids. Sample was excited by spectrofluorometer at wavelength =345 nm and detected from emission wavelength =360 nm. Liquid chromatographic flow rate was 0.15 ml/min for the detection of mono pyrene derivatives (Fig. 3.1-3.2) and '2 µl' injection volume was introduced to LC system from the sample and blank solutions.

A-

B-

C-

Figure 3.1- Fluorescence emission spectra (360 nm), aqueous 64% Acetonitrile was used as mobile phase for the isocratic elution of derivatized acids (100 µg/ml) and 10 times diluted in 50% aqueous THF. A) Blank (without analyte), B) Malonic acid, C) Succinic Acid

D- E- F-

Figure 3.2- Fluorescence emission spectra (360 nm), aqueous 64% Acetonitrile was used as mobile phase for the isocratic elution of derivatized acids (100 µg/ml) and 10 times diluted in 50% aqueous THF ; A) Pthalic Acid, B) Glutaric acid, C) Adipic Acid.

Fig. 3.3 - 3.4 show emission spectra from excimer fluorescence region of derivatized pyrene labeled acids when was excited at wavelength= 345 nm and detected from emission at 480 nm. Liquid chromatographic flow rate was 0.2 ml/min for mono derivatives (Fig. 3.3-3.4). '2 µl' injection volume was injected to LC system from both sample and blank solutions.

A-

B-

C-

Figure 3.3- Fluorescence emission spectra from excimer region (480 nm), aqueous 64% Acetonitrile(0.2 ml/min) was used as mobile phase for the isocratic elution of derivatized acids (100 µg/ml) and 10 times diluted in 50% aqueous THF ; A) blank (without analyte), B) Malonic acid, C) Succinic Acid

D- E- F-

Figure 3.4- Fluorescence emission spectra from excimer region (480 nm), aqueous 64% Acetonitrile was used as mobile phase for the isocratic elution of derivatized acids (100 µg/ml) and 10 times diluted in 50% aqueous THF A) Pthalic Acid, B) Glutaric acid, C) Adipic Acid

3.2- Detection of derivatized compounds on LC-TOF:

TOF mass spectrometer was operated in positive mode of ionization and in full scan mode. . Chromatograms from individual derivatized analytes were given in Fig. 3.5-3.8. Each chromatogram (A-H) in Fig. 3.5-3.8, show at least three or maximum four divisions (windows), upper division represents results from TIC ( total ion chromatogram), all of the lower divisions represent XIC (extracted ion chromatogram) of the ions given in table 2.2

A-

B-

Figure 3.5- Total ion Chromatogram (TIC) and extracted ion chromatogram (XIC) of characteristic ion of derivatized mono and di-pyrene derivatives, analyzed by TOF mass spectrometer while aqueous Methanol was used as mobile phase for the Gradient elution of derivatized acids (100 µg/ml) in DMSO. A) Pthalic Acid, B) Glutaric acid

C-

D-

Figure 3.6- TIC and XIC of characteristic ions of derivatized mono and di-pyrene derivatives, analyzed by TOF mass spectrometer while aqueous Methanol was used as mobile phase for the Gradient elution of derivatized acids (100 µg/ml) in DMSO, C) Succinic Acid; D) Oxalic acid.

E-

F-

Figure 3.7- TIC and XIC of characteristic ions of derivatized mono and di-pyrene derivatives, analyzed by TOF mass spectrometer. Aqueous Methanol was used as mobile phase for the Gradient elution of derivatized acids (100 µg/ml) in DMSO, E) Azelic Acid; D) Pimelic acid.

G-

H-

Figure 3.8- TIC and XIC of characteristic ions of derivatized mono and di-pyrene derivatives, analyzed by TOF mass spectrometer, while aqueous Methanol was used as mobile phase for the Gradient elution of derivatized acids (100 µg/ml) in DMSO, F) Syringic Acid; G) 4-Hydroxy benzoic acid.

4- Discussions:

4.1- Troubleshooting with derivatization reaction

Fluorescence spectrometer was set to the excitation wavelength at 345 nm and emission wavelength at 480 nm during the start of liquid chromatographic experiments. 100 µg/ml (absolute quantity=33.3 µg/ml) multi-component mixture, containing individual analytes (C2-C10) in DMSO were injected with 2 µl Injection volume. Blank solution was same as standard but was without target compounds (C2-C10) in the reaction medium (2.3.1).

Chromatographic peaks were appeared in chromatogram from emission of the compounds eluted from blank solution (PBH, EDC side products) and from standard solutions at 480 nm wavelengths (Fig 3.1-3.3). Chromatographic peaks from all of the analytes were eluted at the same time, which clearly indicates that the target analytes were either not derivatized or eluted within blank solution (main peaks). Main peak in blank was eluted after 5 min, this peak was very broad. Same procedure was done with different concentrations of the target analytes, when series of injections were done in the range of 0.1-1 µg/ml. After injecting different standards with different concentrations there was absence of any separate peak which could be distinguishable from the peaks appeared due to the blank solution.

Multi-component mixture at 3.3 µg/ml concentration were also derivatized according to (section) 2.3.1 scheme, still there was no chromatographic peak which could be related to di-pyrene labeled compounds. Fluorescence emission wavelengths were varied in the range of 460- 490 nm, in order to find proper emission wavelength. Peaks related to analytes were invisible with these experiments. The excimer emission spectra was very broad, as reported elsewhere [1], so there was less possibility that chromatographic peaks from di-pyrene labeled compounds were invisible due to improper wavelength selection by spectroflurometer (typically in the range from 460-490 nm).

After analyzing samples by fluorescence detector these derivatized analytes (proposed) were also injected to TOF mass spectrometer. Table 2.2 was used to select ions at a characteristic m/z, related to each compound in TOF mass spectrometer. The proposed derivatives were not confirmed by TOF mass spectrometer in scan mode. That was an indication of failed derivatization procedure (2.3.1).

Other procedures (2.3.2-2.3.5) were applied in order to verify pyrene derivatives on TOF mass spectrometer and again desired derivatives were absent.

4.2- Confirmation of new pyrene derivatives

Slight vigorous conditions (2.3.6) were applied to confirm derivatives of pyrene, 1M EDC and 10 times concentrated PBH (50mM) were used. As a result of new conditions, chromatographic peaks (C2-C6) from Malonic acid(1.099 min) ,Succinic acid (0.894 min) , Pthalic acid (0.902 min) , Glutaric acid (0.900 min) and adipic Acid(0.900 min) were observed (Fig. 3.3-3.4) after 10 times dilution with 50% aqueous THF. Liquid chromatographic Flow rate was 0.2ml/min and 64% aqueous Acetonitrile was used as mobile phase.

Chromatographic peaks from monomers were in the fluorescence region (360-390 nm) for the same compounds (C2-C6) i.e. those were also present in excimer fluorescence region in aqueous THF solution.

Better resolution and peak symmetry of these of eluting compounds were possible at 360 nm (Fig 3.1-3.2) when comp were within 360-390 nm wavelengths while flow rate was 0.15 ml/min and 64% aqueous Acetonitrile was used as mobile phase. Chromatographic peaks (C2-C6) from monomer fluorescence region from derivatives of Malonic acid (2.02) min), Succinic acid (2.296 min), Pthalic acid (2.372 min), Glutaric acid (2.375 min) and Adipic Acid (2.376 min) were observed (Fig 3.1-3.2), retention time of these compounds were given in parenthesis.

Chromatographic peaks at 480 nm in excimer region which was expected to be from di-pyrene labeled compounds were less intensive comparative to monomeric (360 nm) florescence region. Chromatographic peaks eluted from dicarboxylic acids (C2-C6) derivatives were very different than peaks due to blank solution (Fig 3.1-3.3). Blank solution was run with the same chromatographic and derivatization conditions.

Fluorescence peaks detected from monomer emission (360nm) were intense than excimer fluorescence region (480nm).

All derivatized compounds (C2-C10) that were given in table 2.1 according to scheme 2.3.6 were analyzed again by TOF mass spectrometer. Monomers and dimmers of some of analytes were confirmed through XIC through TOF mass spectrometer (Fig 3.5-3.8). These compounds were analyzed in positive ionization mode and ions selected through a scheme (Fig 1.1).

Aqueous Methanol was used as a mobile phase and analytes were injected to LC-TOF in 100 µg/ml amount. Monomeric pyrene derivatives of Pthalic acid, Glutaric acid, Succinic acid, Oxalic acid, Azelic acid, Pimelic acid, Syringic acid and 4-Hydroxy benzoic acid were confirmed as presented in Figure 3.5-3.8. Dimeric pyrene labeled compounds of Pthalic acid was confirmed clearly; Although Succinic acid and 4-Hydroxy benzoic acid shows small signals corresponding to di-Pyrene labeled compounds.

Monomers of Pthalic acid, Glutaric acid Succinic acid derivatives were confirmed from spectroflurometer as well. Mono derivatives of pyrene were also expected to be fluorescence active. The mono pyrene labeled compounds of Oxalic acid, Azelic acid, Pimelic acid Syringic acid and 4-Hydroxy benzoic acid were not detected by spectroflurometer. The reason of inability of fluorescence detection of these compounds may be attributed to the similar retention time with the peaks from blank solutions (same time elution of the compounds from sample and blank solution) or different excitation or emission wavelengths of these monomers.

Another interesting feature about chromatographic peaks obtained from the excimer fluorescence of resultant derivatives of Malonic acid, Succinic acid, Pthalic acid , Glutaric acid, Adipic Acid (Fig 3.1-3.2) and monomer fluorescence of the same compounds (Fig 3.3-3.4) that were eluted at almost same time. This same retention time of mono and excimer fluorescence of same compounds and result from TOF mass spectrometer indicates that excimer produced were not due to di-Pyrene derivatives but these were due to inter molecular excimer

5- Conclusion:

Present experimental work was focused on fluorescence detection of Compounds containing mono and dicarboxylic acids (C2-C10), these compounds were in pure form were fluorescence inactive. Malonic acid, Pthalic acid, Glutaric acid, Succinic acid and adipic acid compounds were made fluorescence active by the reaction of PBH in presence of EDC. The reaction of PBH with Dicarboxylic acid, to produce di-pyrene derivative, was reported elsewhere [1,2,3] to occur efficiently but in this study only mono pyrene derivatives were shown to produce effectively. In this study of derivatization it was evident that dimeric compounds of dicarboxylic acids were not produced except in case of Succinic acid. Dimeric pyrene derivative of Succinic acid was produced in very low amount. Production of dimeric compound from Succinic acid in small amount certainly was not useful for quantification of the analytes by excimer fluorescence. In contrast to dimeric compounds monomeric compounds from Pthalic acid, Glutaric acid, Succinic acid, Oxalic acid, Azelic acid, Pimelic acid, Syringic acid and 4- Hydroxy benzoic acid were derived by present method (2.3.6) and were confirmed by TOF mass spectrometer. Two derivatives for Malonic acid and adipic acid that were visible by spectroflurometer were not confirmed by TOF mass spectrometer. This study of dervatization also indicates that mono pyrene derivatives of compounds were also useful for detection by spectroflurometer and these compounds can also be separated by reverse phase chromatography using ODS column. This work provides a new method for the sensitive detection of carboxylic acids and this method needs a lot of further effort in order to reach an optimum level for the detection and separation of monomeric or dimeric derivatives of PBH.