Agilent 1290 Infinity Quaternary LC Stepwise Transfer to Methods with MS-Compatible Mobile Phases

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1 Agilent 129 Infinity Quaternary LC Stepwise Transfer to Methods with MS-Compatible Mobile Phases Technical Overview Author A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany Abstract The Agilent 129 Infi nity Quaternary Pump supports fast and convenient method development due to its ability to run ternary and quaternary gradients. This helps to transfer a method containing non-ms compatible mobile phases using nonvolatile buffers to a method using MS compatible mobile phases. The stepwise change from one modifi er to the other permits keeping track of the elution change. This Technical Overview describes how a method, based on phosphate buffer, was changed to a MS compatible method through a stepwise replacement of buffers containing ammonium acetate or formic acid. The developed MS method was transferred to the Agilent 129 Infi nity Binary LC/MS system. 2 1 % phosphate buffer % FA % phosphate buffer % FA

2 Introduction The 129 Infinity Quaternary Pump supports fast and convenient method development due to its ability to run ternary and quaternary gradients. Ternary and quaternary gradients are often used to keep buffer and modifier concentration of a mobile phase mixture constant while using two other channels to run a water/organic gradient. This workflow is extremely helpful for method optimization without preparing several mobile phase sets. The optimum concentration can easily be found, by changing the percentage of the modifier channel from run to run. It is also possible to test different modifiers in one sequence by applying two ternary gradients in series using first, the modifier placed in channel C, and then the modifier placed in channel D. This Technical Overview demonstrates a workflow to transfer a method with mobile phases not compatible with MS into a method with MS-compatible mobile phases. If, for example, a method was developed using NaH 2 PO 4 as buffer, the direct transfer to a MS is not possible. A method compatible with MS needs buffers or modifiers which evaporate in the ion source, such as ammonium acetate or formic acid. Direct injection of the compound mixture onto a LC/MS system using the new modifier implies that the compounds are all known and identification is done through MS spectra. If unknown compounds are present in the compound mixture, the correct correlation between peaks of the LC chromatogram and the MS chromatogram might not be possible, because the elution of the peaks could have changed using the new modifier. Experimental Instrumentation Agilent 129 Infinity Quaternary LC System Agilent 129 Infinity Quaternary Pump Agilent 129 Infinity Autosampler Agilent 129 Infinity Thermostat Agilent 129 Infinity Thermostatted Column Compartment Agilent 129 Infinity Diode Array Detector Agilent 129 Infinity Binary LC/MS System Agilent 129 Infinity Binary Pump Agilent 129 Infinity Autosampler Agilent 129 Infinity Thermostat Agilent 129 Infinity Thermostatted Column Compartment Agilent 129 Infinity Diode Array Detector Agilent 614 Quadrupole Mass Spectrometer Acquisition and Evaluation Software OpenLAB CDS ChemStation version C.1.4 A solution is to stepwise increase the concentration of the MS compatible modifier while, at the same time, the phosphate buffer concentration is decreasing on the 129 Infinity Quaternary UV LC system. By applying a stepwise change, peak tracking is relieved using peak heights or peak areas and UV spectra for identification. Having identified the peak elution difference for the MS compatible modifier, the new method can be passed on to a LC/MS system. Model number G44A G4226A G13B G1316C G4212A Model number G42A G4226A G13B G1316C G4212A G614A 2

3 Chromatographic Conditions Agilent 129 Infinity Quaternary LC Parameter Setting Column Agilent ZORBAX RRHT Eclipse Plus C18, 4.6 mm, 1.8 µm (p/n ) Mobile phases A = water 4% B = methanol 4%, C = mm KH 2 PO 4, ph 7 (adjusted with NaOH) D = mm Ammonium acetate, ph 6.9, or D = 1% formic acid solution, ph 2.1 Gradient 1 4% B to 6% B in utes, at utes 8% B, C=%, D=% Gradient 2 4% B to 6% B in utes, at utes 8% B, C=8%, D=2% Gradient 3 4% B to 6% B in utes, at utes 8% B, C=6%, D=4% Gradient 4 4% B to 6% B in utes, at utes 8% B, C=4%, D=6% Gradient 4% B to 6% B in utes, at utes 8% B, C=2%, D=8% Gradient 6 4% B to 6% B in utes, at utes 8% B, C=%, D=% Flow rate 1.2 ml/ Injection volume µl Column temperature 4 C Detection 24/, Ref 4/8, Hz Transfer from phosphate buffer to ammonium acetate or formic acid solution. Increasing D in 2% steps and decreasing C in 2% steps simultaneously. Chromatographic Conditions Agilent 129 Infinity Binary LC/MS Parameter Setting Column Agilent ZORBAX RRHT Eclipse Plus C18, 2.1 mm, 1.8 µm (p/n ) Mobile phases A = water +.1% formic acid, B = methanol Gradient % B to 6% B in utes, at utes 8% B Flow rate.4 ml/ Injection volume. µl Column temperature C Detection MSD full scan with extracted ion mode Ionization mode API-ES Polarity positive Mass range to 6 Fragmentor 7 Peak width.2 utes 3

4 Analyzed Compounds Tricyclic antidepressants, used as example compounds, were purchased from Sigma Aldrich Chemie GmbH, Germany. The compounds were dissolved in distilled water. H Results and Discussion Due to the structure of these molecules, the separation strongly depends on the ph. The pka values of these compounds are > 9 except for trimipra (pka = 8). Choose a buffer eluent with 2 ph units above or below the pka values of the analytes. The original LC method was based on a phosphate buffer at ph = 7. Ammonium acetate was chosen as MS compatible solvent because the 6.9 ph was close to the ph of the phosphate buffer. Formic acid was used as MS compatible modifier in a second experiment, because it is frequently used for MS analysis. Amitriptyline Imiprae Nortriptyline H 3 C H 3 C H H HCl H Protriptyline Trimiprae, R and S racemate Table 1. Molecular weights and pka values for analyzed compounds. Compound pka value Molecular weight MSD extracted ion Nortriptyline HCl Protriptyline HCl Amitriptyline HCl Trimiprae maleate salt Imiprae HCl

5 Using the 129 Infinity Quaternary Pump, four eluents are deployable. Water was placed in channel A, and methanol was placed in channel B. The eluents containing the modifiers were placed in channels C and D. Channel D contained mm ammonium acetate solution at ph 6.9, or 1% formic acid solution at ph 2.1. The resulting concentration of ammonium acetate in the final experiment was mm, respectively.1% formic acid. To demonstrate the transfer from a non-ms compatible method to a MS compatible method, the following experiments were performed: The 129 Infinity Quaternary Pump was used to analyze tricyclic antidepressant with mm phosphate buffer (channel C) at ph 7. Stepwise addition of ammonium acetate solution (channel D) at ph 6.9. Several methods with decreasing C and increasing D were applied. Six methods had to be set up to go from % C to % D in 2% steps. Stepwise addition of 1% formic acid solution (channel D) at ph 2.1. Several methods with decreasing C and increasing D were applied. Six methods had to be set up to go from % C to % D in 2% steps. Transfer of developed MS method with formic acid to a 129 Infinity Binary LC/MS system Methods were obtained which were compatible with MS. For peak tracking, peak heights and UV-spectra were used Amitriptyline Nortriptyline Protriptyline Trimiprae Imiprae Figure 1. UV-spectra of analyzed compounds, used for identification in addition with peak height.

6 Figures 2 and 3 show an overlay of the six chromatograms with stepwise change of modifiers. Using the ammonium acetate modifier, the retention times shifted to lower values while the elution series remained unchanged. The resolution between the first peaks decreased compared to the chromatogram obtained using the phosphate buffer. Nevertheless, sufficient separation was obtained with ammonium acetate at ph 6.9. The same procedure was repeated with formic acid as the modifier. In this example, the elution series had changed, (Figure 3). All peaks shifted to lower retention times and resolution was significantly reduced. Amitriptylene was now the first peak and nortriptylene the last peak. Protriptylene and trimiprae coeluted and amitriptyline shifted in front of nortriptylene. For identification, the UV spectra were used and the different peak heights. At 6% formic acid, it became obvious that protriptylene and trimiprae will coelute. In a further step, the method was transferred to a column with smaller id and lower flow rates. Typically, MS analysis is done with 2.1-mm id columns and flow rates of about. ml/. 1 % phosphate buffer % NH 4 2% phosphate buffer 8% NH 4 4% phosphate buffer 6% NH 4 1. Protriptylene 2. Trimiprae 3. Nortriptylene 4. Imiprae. Amitriptylene 6% phosphate buffer 4% NH 4 8% phosphate buffer 2% NH 4 % phosphate buffer % NH Figure 2. Separation of antidepressants using ammonium acetate as MS compatible modifier % phosphate buffer % FA 2% phosphate buffer 8% FA 1. Protriptylene 2. Trimiprae 3. Nortriptylene 4. Imiprae. Amitriptylene 6 4% phosphate buffer 6% FA 4 1 6% phosphate buffer 4% FA % phosphate buffer 2% FA % phosphate buffer % FA Figure 3. Separation of antidepressants using formic acid (FA) as MS compatible modifiers. 6

7 The MS method based on formic acid was transferred to a 129 Infinity Binary LC/MS system, (Figure 4). A ZORBAX RRHT Eclipse Plus C18, 2.1 mm, 1.8 µm column was used. The flow was reduced to.4 ml/, the column temperature was raised to C and the injection volume was reduced to. µl. The developed MS method with formic acid provided complete elution of peaks within utes with clear identification TIC Imiprae Trimiprae Amitriptylene Protriptylene Nortriptylene Figure 4. Analys is of tricyclic antidepressants after transfer to the Agilent 129 Infinity Binary LC/MS system using extracted ion chromatograms. Conclusion The transfer of a non-ms compatible method to a method which is applicable to a LC/MS system can be done on an Agilent 129 Infinity Quaternary LC System using a dedicated workflow. The modifier, which is not compatible with MS, is stepwise replaced by a modifier which is usable on a MS. Peak tracking was done using peak height and UV spectra. The stepwise change to the different modifier facilitated the tracking of peaks. At the end, a method was created which was compatible with MS and the elution series was known and could be compared to the primary LC chromatogram. 7

8 This information is subject to change without notice. Agilent Technologies, Inc., 13 Published in the USA, June 1, EN