HPLC amine group detection is useful sensitive and precise for polyamines (biogenic amines), amino acids and other amine groups [1-9]. Traditionally the o-phthaldialdeyde (OPA) solution reagent for amine detection is prepared using borate buffer, pH 10.4, with 50% KOH [9] and it is one of the most sensitive fluorogenic. The OPA technique has been reported to be ten times more sensitive for primary amines, than ninhydrin reaction [9,10]. High sensitive detection is achieved by pre or post column reaction of amine group with OPA- 2 mercaptoethanol complex [10-16]. According to Aminot the borate concentration solution would require excessive pH, with the undesirable risk of precipitation when mixed with sample [12]. However, if boric acid is not completely soluble in the borate solution this methodology should be cause precipitation in the line system, in the cuvette detector and in the tubing line after the “T” connection. If this happens, the entire analysis is compromised. Second Seiler N. [1], a considerably higher concentration of the borate buffer is necessary to ensure alkaline reaction conditions in chromatographic system; so this was an incentive to us to change the borate buffer for alkalinized water, than we have no boric acid at OPA formulation. Pre column derivatization is quite a difficult step that requires exact control of derivatization condition due to poor stability of the adducts formed [11]. In alternative the post column derivatization was choose because ensure prompt reactivity and adducts formation is more suitable when stability problems have to be avoided [11].

Moreover, the prolonged use of boric acid can cause acute or chronic poisoning problems. Acute boric acid poisoning usually occurs when someone swallows powdered roach-killing products that contain borax [2-4]. Chronic poisoning occurs in individuals who are repeatedly exposed to boric acid, i.e., in situations in which they perform laboratory procedures involving amine group detection [4,5]. The fetuses of women exposed to boric acid during pregnancy can lose weight and experience skeletal changes [2,3]. Men who manipulate boric acid for a long time have lower sperm counts and less sperm motility [3]. Consequently, in August 2008, the European Chemical Bureau classified boric acid as toxicity category 2 [5].

The objectives of the present study were to show that:

1- It is feasible specially to eliminate the constant risk of boric acid poisoning using alkalinized pure water instead of boric acid in the preparation of OPA for HPLC amine group reaction and fluorescence detection.

2- Look if the reaction by the two reagents has the same comportment and see if the results will be too close or different for both.

3- We choose three polyamines as standard: putrescine, spermidine and spermine as amine group [9] and human plasma as biogenic amines to check the results of the new OPA solution, comparing with the traditional OPA solution results.

For polyamines separation, an ion-pairing reversed-phase high performance liquid chromatography technique was used, followed by post-column derivatization with o-phthaldialdeyde and fluorescence detection [9].

To fulfill the objective of this study, the polyamines standard of putrescine (PU), spermidine (SD) and spermine (SM) were used as a biogenic amine groups to be evaluated by the OPA methodology, with and without boric acid preparation. Biological matrix (plasma) was used as amine group for both OPA post column reaction and fluorescence detection to compare the reaction activity giving by the alkalinized water versus borate buffer OPA. A considerably higher concentration of the borate buffer is used on traditional OPA preparation to ensure alkaline reaction conditions in chromatographic system [1]; then, the elimination of boric acid and it substitution by water from OPA derivatization solution, will give less chance of substance precipitation in the line system.

Apparatus

The HPLC system from Shimadzu® Corporation, model LC- 10AD, was used with post-column reaction. The system consists of two Shimadzu pumps model LC-10AD, a Shimadzu SIL 10A automatic injector, a Shimadzu CTO-10A oven, a Shimadzu SCL-10A VP gradient mixture unit, and the Shimadzu RF-535 fluorescence detector at 345 nm for excitation and 455 nm for emission. An extra reaction pump for OPA solution was used for post column reaction.

Reagents and buffers

The following gradient solutions were used: Buffer A, 0.10 M sodium acetate and 0.010 M sodium 1-octanesulfonic acid, pH 4.50, with phosphoric acid; Buffer B: 0.20 M sodium acetate and 0.010 M sodium 1-octanesulfonic acid, pH 4.50, with phosphoric acid [9].

The gradient solutions were filtered through Millipore® membrane filters (0.45 μm pore size). The chromatography elution conditions are described in Table 1. The concentrations for the calibration curve were 5 to 150 pmol/injection for PU, 8 to 280 pmol/injection for SD, and 25 to 850 pmol/injection for SM, for both derivatization solutions (Figures 1 and 2).

Figure 1: O-phthaldialdeyde with water calibration curve for amine group detection, pH 10.4, (Y=Area versus X= Concentration, pmol).

Figure 2: O-phthaldialdeyde with borate calibration curve for amine group detection, pH 10.4, (Y=Area versus X= Concentration pmol).

Table 1:Chromatography gradient conditions for o-phthaldialdeydeaminegroup detection.

A new OPA solution was prepared with 0.50 mg 1,2-phthalic dicarboxaldehyde dissolved in 40 mL pure methanol (MeOH), 3 ml Brij 35 (30% solution), and 4 ml 2-mercaptoethanol, and the volume was completed to 250 ml with pure water, pH 10.4, using 50% KOH. The traditional OPA solution was prepared with 0.50 mg 1,2-phthalic dicarboxaldehyde dissolved in 40 ml MeOH, 3 ml Brij 35 (30% solution), and 4 ml 2-mercaptoethanol. The final volume was completed to 250 ml with borate buffer, pH 10.4, using 50% KOH [9-14]. All reagents were HPLC grade with 95-99% purity, purchased from Sigma-Aldrich®. Polyamine standards were stored in a freezer at -5°C. The OPA solutions were stored in amber bottles and pressurized by continuous low flux of helium when in the chromatography system, for analysis. The buffer gradient flow rate was 1.35 ml/min, to give a good polyamines separation and 0.50 ml/min for OPA reagent. The 1,7diaminoheptane (internal standard) was prepared as previously reported by Loser C [9].

Chromatographic method

The column eluate and OPA derivatization reagent (using an extra Shimadzu LC 10 AT pump for the OPA solution) were mixed through a “T” connection. The mixture passed through polypropylene coil tubing (1 m x 0.5 mm i.d.) inside a water bath at 50 ± 1°C, controlled by an automatic thermostat, to improve the post column OPA reaction and facilitate the flow rate [9]. An end-capped 5 μm, 4 × 125 mm, Licrosphere® column, 100 RP-18 (Merck Germany) was used. The temperature reaction bath and the flow rate were adjusted to improve the OPA reaction by amine group. According to Ramadan M.A, [15], the completion reaction of OPA with primary amine was achieved at room temperature (25 ± 2°C) within 10 minutes and 60°C the reaction is affected negatively, causing the instability of the reaction adduct.

The standard solution was a mixture of PU, SD, SM and the internal standard (1.7 diaminoheptane). Biological matrix (plasma) was used as amine group Plasma proteins were precipitated with pure methanol (10 μl plasma and 200 μl MeOH), instead of an acid precipitation as cited by Loser C, then vortex mixed for 40 seconds and centrifuged for 10 min at 4500 rpm. The supernatant was transferred to an Eppendorf tube and evaporated in a heated vacuum centrifugation system (“speed vac.”) for 35 min. The residue was diluted with 100 μl of buffer A, plus the internal standard, vortex mixed, and filtered through a 0.45 μm filter unit (ultrafree- MC, Millipore) by centrifugation for 8 min at 4000 rpm. Next, 10 μl of standard solution or 10 μl of biogenic amine were injected into the HPLC system, running for chromatographic separation.

The standard calibrations curves, (Figure 1, is from OPA borate and Figure 2, is from OPA water), the detection limit (Table 1), the interassay coefficient of variability and the intra-assay precision data were determined to validate this study [6-8]. Statistical analysis was carried out using the STATISTICA 8.0 software (Stat Soft, Tulsa, OK, USA). The t-test for independent samples was used to compare polyamine levels in samples analyzed with and without borate buffer. Values are presented as the mean, standard deviation and the level of significance was set at p<0.05.

The standard calibration curves are illustrated in Figures 1 and 2 and their chromatogram graphic are illustrated at Figures 3 and 4, for borate OPA and water OPA. The correlation factor of water OPA exhibited a linear response and is similar to that of the traditional borate OPA. Besides, the signal of detection sensitivity did not change. Thus, the calibration curves showed a good linearity response for both OPA water (PU, r² = 0.9953; SD, r² = 0.9972; SM, r² = 0.9935) and OPA borate (PU, r² = 0.9960, SD, r² = 0.9956, SM, r² = 0.9925). Figures 3 and 4 refer to standard curve graphic detected with OPA borate and OPA water respectively, and their comportment are the same; independent with reagent is used for reaction.

Figure 3: Chromatogram of biogenic amine standard solution. Peak identities: 1=Putrescene , 2=Internal Standard, 3=Spermidine; After OPA borate reaction.

Figure 4: Chromatogram of biogenic amine standard solution. Peak identities: 1=Putrescene , 2=Internal Standard, 3=Spermidine; After OPA water reaction.

Plasma data (average of triplicate analyses) are presented in Table 2 for both OPA borate and OPA alkalinized water and the standard deviation by the OPA water/OPA borate are less than 0.50. Figures 5 and 6 are showing the chromatogram graphic from plasma sample (biogenic amine), when OPA with borate or OPA with water are used for the reaction detection. There was no difference in the results when the two OPA solutions were compared (p >0.05).

Figure 5: Chromatogram of biogenic amine standard solution. Peak identities: 1=Putrescene , 2=Internal Standard, 3=Spermidine; After OPA water reaction .

Figure 6: Chromatogram of biogenic amine standard solution. Peak identities: 1=Putrescene , 2=Internal Standard, 3=Spermidine; After OPAborate reaction.

Table 2: Concentration of plasma polyamine (amine group) in pmol/ml, using o-phthaldialdeyde with H₂O and borate.Sd by sample group and Sd by OPA group ( Water x Borate).

Recovery data for water OPA were 93% for PU, 91% for SD and 85% for SM, and recovery data for borate OPA were 94, 90 and 86 %, respectively, with no significant difference between preparations. Using water OPA, the limit of detection was 0.10 pmol for PU, 8 pmol for SD and 12 pmol for SM, and, using borate OPA, the limit of detection was 0.11, 6 and 12 pmol for the three polyamines, respectively.

The limit of quantification for water OPA was 0.20 pmol for PU, 15 pmol for SP and 20 pmol for SM, and the limit of detection for borate OPA was 0.22, 13 and 20 pmol for the three polyamines, respectively

The inter- and intra-assay precision data did not differ between the two OPA solutions, with a coefficient of variability of less than 15% and also according to our expectations (Table 3).

Table 3:Inter-assay and intra-day precision for polyamine (amine group) determination with water and borate at pH 10.4

Comparing the calibration curve, the chromatogram graphic for amine detection from the three polyamines using OPA prepared with borate solution and OPA with alkalinized water, the r² values obtained for the two preparations did not differ significantly (p>0.05).

The slope of the calibration curve, precision and inter- and intraday variation for PU, SD and SM were similar for the two OPA solutions (Figure 1-4). Plasma polyamine values also did not differ significantly between the two reagent solutions (p>0.05), showing that the use of the proposed preparation is possible. The results obtained in the present study validated the change of reagent, showing that only the pH is important for the reaction.

The main emphasis of this work and the advantages of using water OPA are the following: a less expensive reagent involving lower or no chance of boric acid crystal precipitation in the tubing line after the “T” connection, or in the cuvette detector, and the absence of risk of human borax poisoning. In addition, the most important thing is that there is less environmental residue contamination by the HPLC detritus. Therefore, the alkalinized water OPA preparation can be used for fluorescence detection of amine groups (as for example, polyamines, using in pre- or post-column reactions, demonstrating that only the alkaline reaction condition (pH 10.4) is important for amine group reaction giving the thiol group .