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Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis


journal homepage: www.elsevier.com/locate/jpba

Short communication

Development of an ICP-MS/MS approach for absolute quantification


and determination of phosphodiester to phosphorothioate ratio in
therapeutic oligonucleotides
Juliusz Bianga, Magali Perez, Damien Mouvet, Caroline Cajot, Philippe De Raeve,
Arnaud Delobel ∗
Quality Assistance sa, Technoparc de Thudinie 2, B-6536 Donstiennes, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phos-
Received 15 December 2019 phorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the
Received in revised form 13 February 2020 determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both
Accepted 14 February 2020
are considered as critical quality attributes and should be determined using robust validated methods.
Available online 15 February 2020
The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous
absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination
Keywords:
of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur)
Absolute quantification
ICP-MS/MS
and optionally determination of sodium (or any other metal) as a counter ion. The performance of the
Mass spectrometry method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model sub-
Oligonucleotide stance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested
Phosphorothioate on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction As for all therapeutic molecules, the determination of their


quantity is critical for both safety and efficacy reasons. Quantifi-
Oligonucleotides are a class of therapeutics that has been under cation can be either absolute or relative. Absolute quantification
clinical development for the past 30 years. Different categories of means that no well-characterized reference standard is required.
oligonucleotides have been developed, among which anti-sense The most commonly used technique for the routine quantification
oligonucleotides (ASO) and aptamers, and more recently small of oligonucleotides in drug substances or drug products is UV spec-
interfering RNA (siRNA) [1]. As of November 2019, 9 oligonu- troscopy [9], but it requires the prior knowledge of the extinction
cleotides were approved for therapeutic use in Europe and/or in coefficient [10]. This coefficient can be estimated by calculation
the US. They have a potential to be used in a wide range of dis- using the base composition of the oligonucleotide, but such an esti-
eases, including cancer, cardiovascular and metabolic conditions, mation has been shown to produce a bias of around 10 %. It can
neurological disorders, and ophthalmic diseases [2–7]. also be determined experimentally by measuring the absorbance
One of the challenges for oligonucleotides used as therapeu- at 260 nm of solutions of known concentrations, but the problem
tics is their pharmacokinetics. In order to make oligonucleotides remains the measurement of this concentration. Another possibil-
amenable to their use as medicines, many chemical modifications ity is to hydrolyze the oligonucleotide with an enzyme and measure
designed to increase resistance against enzymatic digestion have the concentration of released nucleotides by liquid chromatogra-
been developed. Among them, phosphorothioate oligonucleotides phy with UV or MS detection [11]. But this methodology also shows
[8], which include a modification on the phosphate backbone by the limited precision and accuracy. Quantification can also be done
substitution of sulfur for a non-bridging oxygen (see Fig. 1), have using a relative method, such as LC/MS, LC/UV or PCR, but these
shown promise, especially for antisense applications [2]. methods require a well-characterized reference standard.
In phosphorothioate oligonucleotides, the molar ratio of phos-
phodiester to phosphorothioate linkages is also considered as a
critical quality attribute and should be determined experimentally
∗ Corresponding author. during characterization studies. Due to the sequential process, even
E-mail address: arnaud.delobel@quality-assistance.be (A. Delobel). a highly efficient sulfurization process may result in high amounts

https://doi.org/10.1016/j.jpba.2020.113179
0731-7085/© 2020 Elsevier B.V. All rights reserved.
2 J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179

Fig. 1. Structure of RNA and phosphorothioate RNA.

of impurities. A missing substitution may play a role in the potency As sodium is the most common counter-ion for oligonu-
and the in vivo stability of the oligonucleotide versus nucleases cleotides, we also measured its concentration during the same
activity. 31 P-NMR (Nuclear Magnetic Resonance) is the common analytical run, in order to be able to perform mass-balance cal-
way to determine this ratio [12]. This technique requires expen- culations (vide infra).
sive equipment and skilled operators, is relatively slow, sequential We present the preliminary results obtained for the develop-
and lacks sensitivity [13]. Moreover, it cannot be easily imple- ment of an ICP-MS/MS method for the absolute quantification of
mented in a GMP (Good Manufacturing Practices) environment if therapeutic oligonucleotides in drug substances and drug products
this method has to be used for batch release. Another possibil- and for the determination of phosphodiester to phosphorothioate
ity is to use strong-anion exchange chromatography (SAX) with ratio in a single method.
UV detection [14,15]. This technique is more sensitive than NMR
but shows strong limitations in the presence of other process- or 2. Material
product-related impurities.
ICP-MS is mainly used to analyze metals in a wide range of 2.1. Reagents and material
applications, from environmental analysis to quality control of
pharmaceuticals and biopharmaceuticals. However, it can also be ICP/MS-grade standard solutions of sodium, phosphorus and
used in biotechnology and proteomics [16] thanks to its ability to sulfur were obtained from Merck Chemicals. ICP/MS-grade stan-
quantify elements such as phosphorus (for phosphoproteins) or dard solution of yttrium was obtained from VWR International.
sulfur [17,18] (for virtually all proteins). Sulfur determination by Nitric and hydrochloric acid (TraceSELECTTM grade) were obtained
ICP-MS has long been a challenge because of the high ionization from Honeywell Fluka. O,O-Diethyl thiophosphate potassium salt
potential of this element (10.4 eV) and the interferences from poly- (DTP) and ammonium hydroxide solution (28.0–30.0%) were
atomic ions for all sulfur isotopes. The same issue is observed for obtained from Merck Chemicals. Ultrapure deionized water
the determination of phosphorus. (resistivity > 18 M.cm) from a Milli-Q system (Millipore) was
Triple-quadrupole ICP-MS [19] can easily circumvent these used throughout the experiment. Oligonucleotide samples were
issues, with the formation of sulfur and phosphorus oxide cations obtained from Kaneka Eurogentec (Seraing, Belgium).
in the reaction cell, as shown previously by Diez-Fernandez et al.
[20]. 2.2. Sample and standard preparation
Different groups had already mentioned the use of ICP/MS for
the quantification of oligonucleotides, but in other contexts [21,22]. Sample solutions were obtained by gravimetric reconstitution
The ability of triple quadrupole ICP-MS to quantify both phospho- of the oligonucleotide lyophilizates or DPT in water. Then, the
rus and sulfur appeared to us as an opportunity for the absolute solutions were aliquoted into digestion vessels mixed with 0.1 mL
quantification and the efficient determination of phosphodiester of internal standard (50 ppm Yttrium solution), 2.5 mL of concen-
to phosphorothioate ratio in phosphorothioate oligonucleotides, if trated nitric acid and 0.5 mL of concentrated hydrochloric acid and
possible in the same analytical run. covered with caps. Digestion was carried out using a single reaction
Provided the structure of the oligonucleotide is known (which chamber Ultrawave MW digestion system (Milestone Srl, Sorisole
is always the case for oligonucleotide drug substances and drug (BG), Italy), equipped with 15 position sample rack.
products), the phosphorus content can be easily calculated. There- The sample rack with the digestion vessels was installed in
fore, measuring the phosphorus content by ICP-MS/MS can give the reaction chamber containing outer bath (150 mL of water and
access the oligonucleotide concentration, without the need for a 5 mL concentrated nitric acid). The reaction chamber was closed
standard. Only a certified solution of phosphoric acid is needed. and pressurized to 40 bars with compressed nitrogen, heated to
Moreover, by measuring both the phosphorus and the sulfur con- reach 220 ◦ C in 35 min and then the temperature was maintained
tent, the phosphodiester to phosphorothioate ratio can also be at 220 ◦ C for 30 min. After digestion all the solutions for analysis
readily determined. were transferred to polypropylene metal free tubes and diluted to
In an ICP-MS/MS instrument, samples are introduced through 50 mL with water.
a nebulization device, but nebulization and transport efficiency For each digestion cycle at least one procedure blank was
are not equivalent for small and large molecules. As the standard- included.
ization is done using sulfuric acid and phosphoric acid, a very Digested samples were analyzed versus an external five-point
accurate method cannot be obtained by introducing directly the calibration with an internal standardization. Calibration was pre-
oligonucleotide solution in the nebulizer. Therefore, all samples pared by dilution of the reference standards of phosphorus, sulfur
were digested with nitric acid and hydrochloric acid in a microwave and optionally of sodium, in the matrix matched solvent (water
oven, after addition of yttrium as an internal standard. solution of acids at the same level as for digests).
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179 3

Table 1 pound and digested as a sample. Accuracy and precision of the


ICP/MS acquisition parameters.
method were evaluated by digesting different amounts of DTP,
Description Acquisition mode 1 Acquisition mode 2 resulting in solutions containing 0.2 to 0.6 mmol/L of phosphorus.
RF power (W) 1550 1550 Results are presented in Table 2 and Fig. 2. Precision was assessed
Spray chamber Double pass (Scott type) Double pass (Scott type) by calculating the relative standard deviation (RSD) on three deter-
Nebulizer Micromist Micromist minations at each concentration level. RSDs range between 0.2 and
Sample flow (mL/min) 0.4 0.4 0.4 %. Accuracy was assessed by calculating the recovery between
Nebulizer gas (L/min) 0.8 0.8
the theoretical concentration and the one calculated from the stan-
Dilution gas (L/min) 0.2 0.2
Scan mode Mass-shift reaction mode Single quadrupole dardization curve. The mean recoveries at each level range from
Reaction gas O2 (45%) He 99.8–102.4%. The linearity of the method was assessed by plotting
Integ. Time per ion (s) 0.45 0.45 the experimental concentration versus the theoretical concentra-
Acquisition mode Spectrum Spectrum
tion. By linear regression, a slope of 0.989 was obtained, with a
Replicate 3 3
Sweep per replicate 100 100 determination coefficient (R2 ) of 0.9999.
The behavior of DTP during the sample preparation and the
ICP-MS/MS process may be different compared to oligonucleotides,
2.3. ICP-MS/MS analysis but no oligonucleotide with a certified concentration is commer-
cially available. Therefore, to evaluate the accuracy of the method
ICP-MS analysis was performed using an Agilent 8800 ICP-QQQ developed, we analyzed two purified oligonucleotide samples for
triple quadrupole ICP-MS (Agilent Technologies Inc., Santa Clara, which we measured the water content (by coulometric Karl-Fisher
CA, USA) in mass-shift mode using O2 as a reaction gas. For sul- titration) and the phosphorus and sodium content by ICP-MS/MS.
fur quantification, the first quadrupole was set to select 32 S+ (m/z Based on the oligonucleotide content, we were able to calculate
32) and 34 S+ (m/z 34) ions, mass shifted by the reaction with oxy- the expected amount of sodium present as a counter-ion of the
gen in the octapole reaction system and finally detected as 32 S16 O+ oligonucleotide (1:1 molar ratio to phosphorus). The excess sodium
(m/z 48) and 34 S16 O+ (m/z 50) by the second quadrupole. For phos- content was easily calculated, and we assumed that this sodium
phorus quantification, the first quadrupole was set to select 31 P+ was present in the form of sodium chloride (this assumption was
(m/z 31) ion, mass shifted by the reaction with oxygen in the done based information on the synthetic route provided by the sup-
octapole reaction system and finally detected as 31 P16 O+ (m/z 47) plier). The amount of chloride ions was therefore calculated. All
by the second quadrupole. Yttrium was used as internal standard these results are presented in Table 3. A mass balance close to 100
for S and P quantification: the first quadrupole was set to select % was obtained for both samples, which demonstrates indirectly
89 Y+ (m/z 89) ion, mass shifted by the reaction with oxygen in
the excellent accuracy of the method.
the octapole reaction system and finally detected as 89 Y16 O+ (m/z As the method is not intended for the quantification of impu-
105) by the second quadrupole. Sodium determination was car- rities, sensitivity was not assessed, in accordance with ICH Q2(R1)
ried out in single-quadrupole mode; the second quadrupole was guideline on the validation of analytical methods. In order to get
set to select 23 Na+ (m/z 23). Yttrium was used as internal stan- reproducible and accurate results, the method was designed so
dard: the second quadrupole was set to select 89 Y+ (m/z 89). The as the signals measured are far from the detection limits of the
sample introduction system consisted of a quartz Micromist nebu- instrument. The sulfur and phosphorus concentrations measured
lizer, a quartz double path spray chamber and an integral quartz for oligonucleotide samples using the method described here are
injector and torch (Agilent Technologies Inc.). The sample solu- in the ppm range, while limits of quantification are in the low ppb
tions were infused continuously into the spray chamber of ICP-MS range.
by an integrated autosampler I-AS (Agilent Technologies Inc.) and
a peristaltic pump. The operating conditions of the ICP-MS were
optimized and are summarized in Table 1.
3.2. Determination of phosphodiester to phosphorothioate ratio
2.4. Determination of water content in oligonucleotide samples
In order to evaluate the capabilities of the technique to measure
Water content determination was carried by coulometric accurately and precisely sulfur / phosphorus ratios, the instrument
Karl-Fisher titration, with 831 K F Coulometer equipped with was calibrated with diluted NIST solutions of phosphoric and sul-
a Thermoprep device (Metrohm AG, Herisau, Switzerland). An furic acids, varying the sulfur/phosphorus molar ratios from 0 to 1
aliquot of a sample was heated at 180 ◦ C, under dry nitrogen flow at constant phosphorus content. Results are presented in Table 4.
(50 mL/min) which carried the desorbed water from the sample to Accuracy was close to 100 % at all levels tested (with a maximum
the titration cell where after absorption in the electrolyte it was bias of 3%, at low S/P ratio), with excellent precision (%RSD on 3
titrated electrochemically versus an indicator electrode. determinations below 0.6 %). The linearity of the method is pre-
sented in Fig. 3.
3. Results and discussion The performance of the method was studied using a small
molecule standard (O,O-diethyl thiophosphate potassium salt),
3.1. ICP-MS/MS for the analysis of phosphorus and sulfur containing one atom of sulfur and one atom of phosphorus, as well
as samples of therapeutic oligonucleotides (information on sam-
3.1.1. Absolute quantification of oligonucleotides ple characterization provided by the supplier allowed us to know
Absolute quantification is the most straightforward application accurately the phosphodiester to phosphorothioate ratio expected
of ICP-MS/MS for oligonucleotides. By performing a digestion step for each oligonucleotide sample). Results are presented in Table 5.
in the presence of an internal standard before analysis, excellent Very good accuracies (between 99 and 101 %) were obtained for
precision and accuracy can be obtained. In order to evaluate the per- the P O/P = S ratio of all samples with a relative standard deviation
formance of the method, a certified reference standard is required. on 3 determinations below 1%. Even for the most complex sam-
As no oligonucleotide with a certified accurate concentration is ple (oligonucleotide containing 23 bases with 6 phosphorothioate
commercially available, a more characterized sample, O,O-diethyl linkages and a cholesteryl group), an accuracy of 99 % is obtained,
thiophosphate potassium salt (DTP) was taken as a model com- with a relative standard deviation on 3 determinations of 0.3 %.
4 J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179

Table 2
Results obtained for the evaluation of absolute quantification method performance.

Theoretical DTP Replicate Phosphorus concentration Accuracy (expressed as % Average accuracy (%) RSD% (n = 3)
concentration (mmol/L) measured (mmol/L) recovery)

1 0.198 102.9
0.192 2 0.196 102.0 102.4 0.4
3 0.197 102.3
1 0.297 100.9
0.295 2 0.299 101.3 101.2 0.2
3 0.298 101.2
1 0.387 100.7
0.384 2 0.386 100.6 100.7 0.2
3 0.388 100.9
1 0.475 99.8
0.475 2 0.477 100.2 99.8 0.4
3 0.473 99.4
1 0.579 100.3
0.578 2 0.581 100.6 100.3 0.3
3 0.577 99.9

Fig. 2. Evaluation of method linearity for DTP absolute quantification based on phosphorus content.

Table 3
Evaluation of method accuracy based on phosphorus, sodium, and water content on two purified oligonucleotide samples.

% w/w in sample powder


Sample Mass balance (%)
Oligonucleotide Sodium (total) Water Excess sodium (calc.) Chloride (calc.)

Oligonucleotide 1 30.0 28.1 3.5 26.0 40.1 101.7


Oligonucleotide 2 63.2 14.6 6.6 10.1 15.6 100.1

Table 4
Results obtained for the evaluation method performance for the determination of phosphodiester to phosphorothioate ratio.

Measured conc. P/S molar ratio Average


Test solution Replicate Accuracy % RSD %
(mmol/L) Accuracy %

P S Theoretical Experimental

1 0.587 0.626 0.932 0.937 100.5


T1 2 0.589 0.628 0.932 0.938 100.6 100.6 0.06
3 0.588 0.627 0.932 0.938 100.6
1 0.583 0.184 3.108 3.159 101.6
T2 2 0.582 0.186 3.108 3.122 100.4 101.1 0.60
3 0.580 0.184 3.108 3.142 101.1
1 0.581 0.061 9.326 9.470 101.5
T3 2 0.579 0.061 9.326 9.421 101.0 101.3 0.26
3 0.578 0.061 9.326 9.428 101.1
1 0.579 0.012 46.630 45.553 97.7
T4 2 0.575 0.012 46.630 45.127 96.8 97.3 0.49
3 0.576 0.012 46.630 45.485 97.5
J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179 5

Fig. 3. Evaluation of method linearity for phosphodiester to phosphorothioate ratio determination.

Table 5
Evaluation of the accuracy of phosphodiester to phosphorothioate ratio determination of DTP and oligonucleotide samples.

Sample Number of Number of Theoretical P/S Experimental Accuracy % RSD (n = 3)


P/mol S/mol ratio P/S ratio

O,O-diethyl thiophosphate 1 1 1.000 0.9913 99.1 0.1


Oligonucleotide 1 (20 mer, full 2 -OMe full PS) 19 19 1.0000 1.010 101.0 0.4
Oligonucleotide 2 (20 mer, full PS) 19 19 1.0000 0.9999 100.0 0.4
Oligonucleotide 3 (20 mer, full 2 -OMe full PS) 19 19 1.0000 1.0032 99.7 0.3
Oligonucleotide 4 (22 mer, full PS) 21 21 1.0000 0.9884 101.2 0.3
Oligonucleotide 5 (22 mer, full PS) 21 21 1.0000 0.9935 100.7 0.8
Oligonucleotide 6 (23 mer, full 2 -OMe, 6 PS, 5 -Cholesteryl) 23 6 3.833 3.876 101.1 0.3

4. Conclusion CRediT authorship contribution statement

We developed an ICP-MS/MS method based on the quantifica- Juliusz Bianga: Methodology, Investigation, Formal analysis,
tion of phosphorus and sulfur after microwave digestion for the Writing - original draft. Magali Perez: Supervision. Damien Mou-
absolute quantification of oligonucleotides (based on phosphorus vet: Supervision. Caroline Cajot: Resources. Philippe De Raeve:
content), and for the determination of phosphodiester to phospho- Conceptualization, Methodology, Supervision. Arnaud Delobel:
rothioate ratio in purified therapeutic oligonucleotide samples, in Project administration, Writing - review & editing.
the same analytical run. Although the quantification of phospho-
rus and sulfur by ICP-MS/MS was previously described, this is to
the best of our knowledge the first application of this technology Declaration of Competing Interest
to therapeutic phosphorothioate oligonucleotides for both abso-
lute quantification and phosphodiester-to-phosphorothioate ratio The authors declare that they have no known competing finan-
determination. Around 10 mg of sample are required to perform the cial interests or personal relationships that could have appeared to
test, and results can be obtained within one day of analytical work, influence the work reported in this paper.
including sample preparation. Even if additional investigations may
be needed, preliminary results are very encouraging, with excel-
Acknowledgments
lent precision and accuracy obtained on reference standards and
therapeutic oligonucleotide samples.
The authors would like to thank Kaneka Eurogentec for provid-
As there is no chromatographic separation prior to ICP-MS/MS
ing oligonucleotide samples.
analysis, this method is intended to be applied to highly-purified
therapeutic-grade oligonucleotides. The absolute quantification
method will actually quantify the oligonucleotide and all the References
potential impurities, while another purity method (usually a chro-
matographic method with optical or MS detection) may be used to [1] C.A. Stein, D. Castanotto, FDA-approved oligonucleotide therapies in 2017,
Mol. Ther. 25 (2017) 1069–1075, http://dx.doi.org/10.1016/j.ymthe.2017.03.
determine the purity profile. The method could be used in routine 023, PMID - 28366767.
testing of oligonucleotides in a GMP environment, as an orthogonal [2] C.F. Bennett, Therapeutic antisense oligonucleotides are coming of age, Annu.
method to 31 P-NMR. Rev. Med. 70 (2019) 307–321, http://dx.doi.org/10.1146/annurev-med-
041217-010829, PMID - 30691367.
6 J. Bianga, M. Perez, D. Mouvet et al. / Journal of Pharmaceutical and Biomedical Analysis 184 (2020) 113179

[3] L. Echevarría, P. Aupy, A. Goyenvalle, Exon-skipping advances for Duchenne Chromatogr. A 599 (1992) 35–42, http://dx.doi.org/10.1016/0021-
muscular dystrophy, Hum. Mol. Genet. 27 (2018) R163–R172, http://dx.doi. 9673(92)85456-4.
org/10.1093/hmg/ddy171, PMID - 29771317. [15] V. Metelev, S. Agrawal, Ion-exchange high-performance liquid
[4] C.I.E. Smith, R. Zain, Therapeutic oligonucleotides: state of the art, Annu. Rev. chromatography analysis of oligodeoxyribonucleotide phosphorothioates,
Pharmacol. Toxicol. 59 (2018) 1–26, http://dx.doi.org/10.1146/annurev- Anal. Biochem. 200 (1992) 342–346, http://dx.doi.org/10.1016/0003-
pharmtox-010818-021050, PMID - 30285540. 2697(92)90476-n, PMID - 1632499.
[5] O. Khorkova, C. Wahlestedt, Oligonucleotide therapies for disorders of the [16] O. Chahrour, J. Malone, Inductively coupled plasma mass spectrometry
nervous system, Nat. Biotechnol. 35 (2017) 249–263, http://dx.doi.org/10. (ICP-MS) applications in quantitative proteomics, Protein Pept. Lett. 23 (2016)
1038/nbt.3784, PMID - 28244991. 1, http://dx.doi.org/10.2174/0929866523666161213094936, PMID -
[6] K.E. Lundin, O. Gissberg, C.I.E. Smith, Oligonucleotide therapies: the past and 27964701.
the present, Hum. Gene Ther. 26 (2015) 475–485, http://dx.doi.org/10.1089/ [17] H.-S. Lee, S.H. Kim, J.-S. Jeong, Y.-M. Lee, Y.-H. Yim, Sulfur-based absolute
hum.2015.070, PMID - 26160334. quantification of proteins using isotope dilution inductively coupled plasma
[7] K. Takakura, A. Kawamura, Y. Torisu, S. Koido, N. Yahagi, M. Saruta, The mass spectrometry, Metrologia 52 (2015) 619–627, http://dx.doi.org/10.
clinical potential of oligonucleotide therapeutics against pancreatic cancer, 1088/0026-1394/52/5/619.
Int. J. Mol. Sci. 20 (2019) 3331, http://dx.doi.org/10.3390/ijms20133331, PMID [18] L. Cid-Barrio, F. Calderón-Celis, P. Abásolo-Linares, M.L. Fernández-Sánchez,
- 31284594. J.M. Costa-Fernández, J.R. Encinar, A. Sanz-Medel, Advances in absolute
[8] F. Eckstein, Phosphorothioates, essential components of therapeutic protein quantification and quantitative protein mapping using ICP-MS, TrAC
oligonucleotides, Nucleic Acid Ther. 24 (2014) 374–387, http://dx.doi.org/10. Trends Anal. Chem. 104 (2018) 148–159, http://dx.doi.org/10.1016/j.trac.
1089/nat.2014.0506, PMID - 25353652. 2017.09.024.
[9] D. Michaud, Oligonucleotide assay and potency, in: Handb. Anal. [19] L. Balcaen, E. Bolea-Fernandez, M. Resano, F. Vanhaecke, Inductively coupled
Oligonucleotides Relat. Prod., CRC Press, 2011, pp. 285–305, http://dx.doi.org/ plasma – tandem mass spectrometry (ICP-MS/MS): a powerful and universal
10.1201/b10714-10. tool for the interference-free determination of (ultra)trace elements – a
[10] V. Murugaiah, Determination of extinction coefficient, in: Handb. Anal. tutorial review, Anal. Chim. Acta 894 (2015) 7–19, http://dx.doi.org/10.1016/j.
Oligonucleotides Relat. Prod., CRC Press, 2011, pp. 351–359, http://dx.doi.org/ aca.2015.08.053.
10.1201/b10714-13. [20] S.D. Fernández, N. Sugishama, J.R. Encinar, A. Sanz-Medel, Triple quad ICPMS
[11] H. Aygün, Determination of Base composition, in: Handb. Anal. (ICPQQQ) as a new tool for absolute quantitative proteomics and
Oligonucleotides Relat. Prod, CRC Press, 2011, pp. 439–452, http://dx.doi.org/ phosphoproteomics, Anal. Chem. 84 (2012) 5851–5857, http://dx.doi.org/10.
10.1201/b10714-18. 1021/ac3009516.
[12] B.L. Hirschbein, K.L. Fearon, 31P NMR spectroscopy in oligonucleotide [21] Q. Tu, E.N. Guidry, F. Meng, T. Wang, X. Gong, A high-throughput flow
research and development, Antisense Nucleic Acid Drug Dev. 7 (1997) 55–61, injection inductively coupled plasma mass spectrometry method for
http://dx.doi.org/10.1089/oli.1.1997.7.55, PMID - 9055040. quantification of oligonucleotides, Microchem. J. 124 (2016) 668–674, http://
[13] M. DeRider, D. Brooks, G. Burt, Structural Determination by NMR, Handb. dx.doi.org/10.1016/j.microc.2015.10.011.
Anal. Oligonucleotides Relat. Prod., 2011, pp. 361–384, http://dx.doi.org/10. [22] S. Studzińska, S. Mounicou, J. Szpunar, R. Łobiński, B. Buszewski, New
1201/b10714-14. approach to the determination phosphorothioate oligonucleotides by ultra
[14] B.J. Bergot, W. Egan, Separation of synthetic phosphorothioate high performance liquid chromatography coupled with inductively coupled
oligodeoxynucleotides from their oxygenated (phosphodiester) defect species plasma mass spectrometry, Anal. Chim. Acta 855 (2015) 13–20, http://dx.doi.
by strong-anion-exchange high-performance liquid chromatography, J. org/10.1016/j.aca.2014.12.010, PMID - 25542085.

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