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BIOANALYSISIdentification of an unknown medicine in
serum through liquid-liquid extraction and
solid phase extraction
Tymo Zwakenberg S4326253
30-06-2023
ABSTRACT
Liquid-liquid extraction (LLE) and solid phase extraction (SPE) are essential techniques in analytical
chemistry for the separation and purification. In this experiment, it was tried to identify an unknown
medicine sample and evaluate the extraction results by means of statistical analysis. The experiment
consists of a mixed-mode solid phase, combining a hydrophobic portion and a cation-exchange
portion. The drug was identified as oxazepam based on pH measurements, extraction patterns, and
comparison with known drugs. Confirmation was obtained through the use of an internal standard
and recovery calculation, which indicated a concentration recovery rate of 100.03%-104.50%.
Statistical analysis was performed to determine the relationship between the concentration of the
unknown drug (X) and the response (Y). The regression analysis showed a significant regression
relationship. Similarly, the confidence interval and hypothesis tests for the intercept (α) indicated that
the line did not pass through the origin. The goodness of fit test showed that the regression model
adequately explains the variability in the response variable. The coefficient of determination (R-
squared) indicated that 99.162% of the variance in Y can be explained by the regression model,
indicating a strong relationship between X and Y. However, the lack of fit test showed the possibility
of a nonlinear relationship or the presence of other variables.
Overall, this experiment demonstrated the successful application of LLE and SPE techniques for the
extraction and identification of an unknown medicine sample. The statistical analysis provided
valuable insights into the relationship between the concentration of the unknown drug and the
response, highlighting the significance of the regression model with some contradictions.
INTRODUCTION experimental setup variabilities which will be
Liquid-liquid extraction (LLE) is a fundamental shortly discussed.
principle in analytical chemistry to separate The hydrophobicity determines whether the
molecules based on their distribution molecule remains in the aqueous phase or is
between two immiscible liquid phases. It is extracted into the organic phase. To achieve
used in bioanalysis to enrich and purify small enrichment in the organic phase, the
molecule pharmaceuticals from complex distribution coefficient (P) of the compound
biological samples. LLE relies on exploiting must be higher than 1, while interfering matrix
the differences in Physico-chemical properties compounds should have P values less than 1.
that affect the distribution of target molecules Additionally, the choice of organic solvents
between a hydrophilic, aqueous phase and a should be considered for LLE. Solvents with
hydrophobic organic phase. lower densities than water to form the upper
The success of LLE depends on the phase, while those with higher densities form
hydrophobicity of the target molecule and the lower phase. To maximize extraction yield,
the hydrophobicity of the organic solvent, as
Date submitted, Protein Analysis or Medicine Analysis | 1
, BIOANALYSIS
indicated by its octanol-water distribution that can interact with analytes and their
coefficient (logP), is an important factor. One instability at high pH values. Polymeric
of the most important factors is adjusting the materials have gained popularity as they offer
pH of the aqueous phase. This is an effective stability across a wider pH range, but they
strategy for enhancing extraction yield, require careful surface modification to reduce
especially for pharmaceutical compounds their inherent hydrophobicity. Therefore, the
containing basic and/or acidic groups. most often used SPE materials can be
Protonated bases or deprotonated acids are categorized into normal phase, reversed
more hydrophilic and can be selectively phase, ion-exchange, and mixed-mode,
extracted by controlling the pH based on their offering various selectivity options. When
dissociation constant. The extraction influence performing SPE, it is crucial to assess the
of the pH is represented into the following properties of the analyte, such as its charge,
equations: hydrophobicity, and potential for hydrogen
bonding or aromatic interactions.
𝑃⋅𝑉 Additionally, conditioning the SPE material
𝐹𝑜𝑟𝑔 = 𝐾 for acidic compounds.
𝑃⋅𝑉 +1 + 𝑎+ before use is essential, including equilibration
[𝐻 ]
for ion-exchangers at the desired pH and salt
𝑃⋅𝑉 concentration, and conditioning with a water-
𝐹𝑜𝑟𝑔 = [𝐻+]For basic compounds.
miscible organic solvent for hydrophobic
𝑃⋅𝑉 +1 + 𝐾
𝑎
reversed-phase materials.
Ultimately, the addition of salt to the aqueous
In this practical experiment, a mixed-mode
phase increases its ionic strength, leading to a
column will be used as the solid phase, which
phenomenon called "salting out" where
combines a hydrophobic portion and a cation-
organic molecules are pushed out of the
exchange portion. The hydrophobic portion
aqueous solution. Adding salt also increases
consists of an eight-carbon chain (C8), while
the density of the aqueous phase, which could
the cation-exchange portion contains a
be beneficial when working with organic
negatively charged propylbenzenesulfonate
solvents of lower density. At last, it helps to
(SCX) group that specifically binds to
prevent the formation of emulsions at the
positively charged analytes. The retention
interface between the two liquid phases [1,
mechanism of the analytes on the column
2&3].
depends on their pH-dependent properties
[1,2 &3]. The goals of the experiment are to
An additional or different way of extracting
get acquainted with different extraction
the wanted compound is solid phase
extraction (SPE). methods by performing an identification of
The SPE procedure consists of four main unknown medicine sample 3, which was
steps: provided, and to evaluate the results of the
1. conditioning the solid-phase material to extraction through statistical analysis. The
retain the analyte while minimizing retention identity of the unknown medicine can have 6
of matrix components. different options, either an NSAID,
2. loading the solid phase with the target benzodiazepine or a TCA, with 2 possibilities
sample. for each category [7&8].
3. washing the solid phase to remove matrix
components while retaining the analyte.
4. eluting the analyte from the solid phase for MATERIALS & METHODS
further analysis. Liquid-liquid extraction
Sample preparation
The eluted analyte is evaporated to dissolve it An unknown medicine dilution stock of 20
in a useful solvent for the analysis such as LC- µg/ml was prepared. Three glass (A, B & N)
MS. The choice of SPE material depends on vials were subsequently prepared with the
the properties of the analyte. Silica-based following composition: A: 800 µl serum, 1600
materials have been widely used due to their µl diluted medicine, and 1400 µl demi-water.
availability and surface chemistries, but they B: 800 µl serum, 1600 µl diluted medicine, and
have limitations such as residual silanol groups 1400 µl demi-water. N: 800 µl serum, 1600 µl
Date submitted, Protein Analysis or Medicine Analysis | 2
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