Evaluation of basic analytes using a novel hybrid reversed phase method for solid phase extraction by HPLC-MS

The Microlute™ CP range from Porvair Sciences, offers a new method of solid phase extraction (SPE) using a unique hybrid polymer structure combined with retentive media. This composite design enhances the flow-through of samples throughout its porous structure to maximise interactions between analytes and the solid phase to deliver a highly reproducible SPE method. This application note demonstrates the robust SPE LC-MS methodology of the Microlute™ CP Reversed Phase (RP) SPE 30 mg plates on basic analytes by comparing recoveries and reproducibility benefits over loose-filled products.

Introduction

Solid phase extraction (SPE) is a sample preparation method for the clean-up of samples before analysis with HPLC or GC analysis. SPE offers a number of advantages to the analyst, including less system downtime and troubleshooting, cleaner chromatograms with a reduction of contaminating compounds, and more reproducible analyte recoveries. Traditionally, SPE methods use loose-filled resins which can create problems, such as voids in the sorbent beds leading to channelling, inconsistent flow-through of solutions, instability at extreme pHs and residual silanol activity. This materialises in less interactions between analytes with the active resin leading to inconsistent results and poor analyte recovery.
Over more recent years, significant advantages have been seen from using polymer-based materials.  The processes used to synthesise polymer-based sorbents enable incorporation of numerous chemical functionalities into the porous framework (Figure 1). The ability to generate highly specific and regular functionality gives high retention capacity for different types of compounds including basic compounds and stability at extreme pHs.[1]
With only 20% of pharmaceutical analytes exhibiting acidic properties, basic analytes accounts for 70% of pharmaceutical compounds making them the most abundant analyte type being analysed via HPLC/GC. Unlike neutral and acidic compounds, basic compounds require less method development to ensure they are sufficiently retained on the sorbent medium.[2]

Retention of basic analytes from polar solutions onto reversed phase SPE materials occurs due to Van der Waal forces or dispersion forces between carbon-hydrogen bonds of the analyte and the functional groups bonded to the sorbent material. Stronger Van der Waal forces lead to greater retention on the reversed phase. Finally, silica-based resins can be sensitive to stationary phase collapse if the sorbent bed becomes dry after the condition step whereas, polymer-based sorbents are less susceptible to drying out. This enables quicker SPE method development and gives the analyst ease of mind when performing SPE. In addition, minor packing differences of sorbents into cartridges and wells can cause significant differences in the flow of solutions when performing SPE steps.
The outlined advantages of polymer-based sorbents greatly suggest a superior method of solid phase extraction not only in performance but increased reproducibility of recovery and retention of analytes of interest. To demonstrate the sensitivity and robustness of the 30 mg Microlute™ CP RP, an SPE experiment was performed using spiked aqueous sample matrices containing basic analytes and was compared against five commercially available 30 mg loose-filled reversed phase products.

Evaluation of Basic Analyte Recovery and Reproducibility

To highlight the sensitivity of the 30 mg RP composite, SPE was carried out on 12 wells using spiked aqueous sample matrices with eight basic analytes. 10 µg of each analyte was loaded onto each well, eluted with organic solvent, dried, and reconstituted before being diluted ready for analysis with LC-MS. The same technique was used to compare performance of equivalent competitor 30 mg plates.

Experimental

Chemicals:
Caffeine, atenolol, salbutamol, propranolol, nortriptyline, protriptyline, imipramine, desipramine, amitriptyline, formic acid, methanol, water, 35% ammonia solution.

Sample Preparation:
A stock of 1,000 µg/ml for each of the basic analytes was made in methanol. A basic load solution was made by using 500 µl stock solution and diluting to 50 ml with water containing 0.1% (v/v) ammonia solution.

Solid Phase Extraction Method:
Figure 2. Flow schematic of solid phase extraction method for basic analytes.
Table 1. LC system conditions for chromatographic separation of basic analytes.
LC Conditions:
LC system    Agilent LC-MS, consisting of a 1260 LC and Single Quadrupole Mass Spectrometer.
Column    Raptor Biphenyl 30 x 2.1 mm, 1.8 µm
Column temp.    45°C
Injection volume    2.00 µL
Flow rate    600 µL/min
Mobile phase A    0.1% Formic acid in water
Mobile phase B    0.1% Formic acid in methanol

Solvent Composition

Table 2. Mass spectrometer conditions.
Parameter    Value
Gas Temperature    350 ºC
Gas Flow    13 L/min
Nebuliser    30 psi
Capillary Voltage    4000 V
Fragmentor Voltage    100 V
Scan Type    SIM
Ion Mode    ESI

The dwell time (ms) varied depending on compound detection as followed:
Table 3. Dwell time values for basic analytes.
Compound    Dwell Time (ms)

Atenolol    70
Salbutamol    70
Caffeine    150
Propranolol    130
Imipramine    50
Amitriptyline    50
Desipramine    50
Protriptyline    50
Nortriptyline    50

Results and Discussion

Chromatogram

Figure 3. Chromatogram of basic analytes calibration standard. Peak assignments can be found in Table 4.

Peak Assignment

Table 4. Properties and retention times for the basic compounds analysed - a Predicted value from Pubchem [3].
Number    Compound    Type    R.T
(min)    Formula    Molecular Mass    LogP[a]    pKa[a]
1    Salbutamol    Basic    0.65    C13H21NO3    239.31    0.3    10.3
2    Atenolol    Basic    1.27    C14H22N2O3    266.34     0.2    10.4
3    Caffeine    ISTD    3.89    C8H10N4O2    194.19    -0.1    14.0
4    Propranolol    Basic    5.55    C16H21NO2    259.34    3.0    9.4
5    Desipramine    Basic    6.80    C18H22N2    266.4    4.9    9.6
6    Protriptyline    Basic    7.00    C19H21N    263.4    4.4    9.7
7    Imipramine    Basic    7.16    C19H24N2    280.4    4.8    9.4
8    Nortriptyline    Basic    7.41    C19H21N    263.4    3.9    10.5
9    Amitriptyline    Basic    7.57    C20H23N    277.4    5.0    9.4

Recovery comparisons against competitor products

Recovery is an important metric in any sample preparation method. Higher recoveries allow more sensitive methods along with lower limits of quantification and detection. The data shown in Figure 4 highlights how the Microlute™ CP Reversed Phase product can give very high recoveries for basic analytes.
Significantly higher recoveries of hydrophobic basic analytes can be seen when compared to competitor plates. For example, the recovery of amitriptyline for the Porvair MicroluteTM product is 91.6%, comparing this value to the best performing competitor (competitor 3) at 69.6%. This shows an increase in recovery of 31.6%. An increase in recovery of 56.3% can be seen when comparing this recovery against the worst performing competitor.
It might be thought that such an increase in recoveries of more hydrophobic bases would result in a decrease in recoveries for the more hydrophilic bases. From the data collected, this is shown not to be the case. Instead there is a great balance in recoveries of both hydrophilic bases and hydrophobic bases. The Porvair MicroluteTM CP RP maintains very high recoveries for the full range of basic compounds.
Figure 5. Basic analyte recovery %RSD comparisons against equivalent competitor SPE products. Number of wells tested = 12.
The basic analytes %RSD values can be seen Figure 5. The Porvair MicroluteTM CP RP obtains %RSD values of less than 3.3% for every compound. This on average beats all competitors except competitor 4. However, when you consolidate both recovery and reproducibility metrics the Porvair MicroluteTM CP RP combines better recoveries with great reproducibility values to produce the best all-around results for the recovery of basic compounds.

Conclusion

The MicroluteTM CP RP 30 mg 96 well plate can selectively retain and elute basic compounds. The %RSD values are on average significantly lower than competitors, giving more reproducible results. The hybrid technology ensures even liquid flow rates throughout the SPE process, which leads to sufficient time for Van der Waals forces of interaction to take place between the sorbent and the analytes. No analyte is lost in the load step of the SPE process leading to high recovery values for all basic compounds. The MicroluteTM CP RP 96 well-plate for solid phase extractions offers significant benefits for the recovery of hydrophobic basic analytes, as well as keeping %RSD values <5%.  

References

1. Poole, C., 2019. Solid-phase extraction. 1st ed. Elsevier, Chapter 3 - Porous polymer sorbents.
2. R. Veigure, K. Lossmann and M. Hecht et al. / Journal of Chromatography A 1613.
3. Pubchem. [online] Available at: PubChem (nih.gov)