ApplicationNews

No.L447

High Performance Liquid Chromatography

Analysis of Compounds Containing a Phosphate Group Using the New MastroTM High Pressure-Resistance Stainless Steel-Free LC Column

LAAN-A-LC-E225

Substances containing phosphate groups or metal chelates are often found in pharmaceuticals and metabolites. When subjected to HPLC analysis, these compounds are known to adsorb to metallic surfaces in the HPLC system flow line, thereby causing adverse peak tailing in their chromatograms. The typical remedy for this has been to add chelating agents and salts to the mobile phase, but the use of such nonvolatile eluents is not applicable when using an LC/MS as the detector.Here, we examined the effects of different flow line mater ia l s on the peak shape of coord inat ion compounds. Also, using a formic acid mobile phase, which is commonly used in LC/MS analysis, we examined the inhibiting effect on metal adsorption of coordination compounds that is provided with MastroTM high pressure-resistance stainless steel-free analytical column.

n Influence of HPLC Tubing MaterialWhen using mobile phases applicable to LC/MS analysis, adsorption to stainless steel tubing of any coordination compound that may be present can result in peak tailing. Thus, using a mobile phase applicable to LC/MS, together with a conventional column, we investigated the effect of the material used for the autosampler outlet tubing (600 mm) on peak shape in the measurement of a nucleotide standard solution. Stainless steel and PEEK materials were both used for the tubing.A nucleotide is a compound consisting of a nucleoside that contains one or more phosphate groups. ATP (adenosine triphosphate) is a compound that contains three such phosphate groups (Fig. 1). We prepared solutions of ATP and AMP (approximately 10 mg/L each) and conducted analysis using the analytical conditions shown in Table 1.When using stainless steel tubing, the delay in retention time due to adsorption was verified, but regardless of the tubing material used, no significant difference in tailing due to adsorption was observed. It is therefore presumed that the column plays a larger role in the occurrence of peak tailing.

Fig. 1 Structural Formulas of Nucleotides

Table 1 Analytical Conditions

Column : Typical C18 (100 mm L. × 2.1 mm I.D., 3 μm)Instrument : NexeraMobile Phase : 10 mmol/L Ammonium Formate in Water:Acetonitrile=99:1Flowrate : 0.2 mL/minColumn Temp. : 40 °CDetection : SPD-M20A at (254 nm)Injection Vol. : 1 μL

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min

2

1

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min

2

1

■Peaks1. Adenosine Triphosphate2. Adenosine Monophosphate

Fig. 2 Difference in Peak Shape Due to Tube Material (Upper: PEEK Material, Lower: Stainless Steel)

Adenosine monophosphateAdenosine triphosphate

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© Shimadzu Corporation, 2013www.shimadzu.com/an/

ApplicationNews

No.

First Edition: May. 2013

L447

Next, we investigated the effect of the MastroTM stainless steel-free column on retention time shift and peak tailing. The high pressure-resistance stainless steel-free MastroTM

column consists of a stainless steel body with a polymer-coated inner surface, and a frit which adopts a highly cross-linked polymer material. As metallic activity is reduced to the greatest extent possible, adsorption in the column of substances susceptible to metal coordination is greatly suppressed (Fig. 3).The main component of an adrenocorticotropic hormone preparation is hydrocortisone sodium phosphate, which contains one phosphate group. On the other hand, the main component of the water-soluble hydrocortisone preparation is hydrocortisone sodium succinate, which does not contain a phosphate group (Fig. 4).We conducted analysis of these compounds using a mobile phase that is applicable to LC/MS. Table 2 shows the analytical conditions.With a typical C18 column, tailing is generally seen in analysis of hydrocortisone sodium phosphate, with delayed retention t ime and peak reversal due to adsorption. With the MastroTM column, however, sharp peak shapes were obtained for both substances (Fig. 5).Thus, we confirmed that the column plays a large role in the suppress ion of peak ta i l ing of coordinat ion compounds when using a mobile phase applicable to LC/MS, and that a stainless steel-free column is effective in this respect.

n Hydrocortisone Sodium Phosphate and Hydrocortisone Sodium Succinate Analysis

Fig. 4 Structure of Hydrocortisone

Fig. 3 MastroTM Column Image

Table 2 Analytical Conditions

Column : MastroTM C18 (100 mm L. × 2.1 mm I.D., 3 μm)Typical C18 (100 mm L. × 2.1 mm I.D., 3 μm)

Instrument : NexeraMobile Phase : 0.1 % Formic Acid in Water /0.1 % Formic Acid in

Acetonitrile = 50:50 Flowrate : 0.2 mL/minColumn Temp. : 40 °C Detection : SPD-M20A at (254 nm)Injection Vol. : 1 μL

PO3Na2

1.00 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

1

2

MastroTM C18カラム

Typical C18カラム

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min0

2

1

■Peaks1. Hydrocortisone Sodium Phosphate2. Hydrocortisone Sodium Succinate

Fig. 5 Chromatograms of Hydrocortisone Sodium Phosphate and Hydrocortisone Sodium

The data and examples of analysis of the adrenocorticotropic hormone and aqueous hydrocortisone preparations were obtained in collaboration with Associate Professor Kenichiro Todoroki, Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka.

For more information about the MastroTM column, please contact Shimadzu GLC Ltd.

Polymer frit

Polymer liningStainless steel Body

Stationary phase