Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C₁₈ column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one-dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.

二维液相色谱-质谱联用技术（2D-LC / MS）已成功用于多种生物制药应用，但对寡核苷酸分析的应用却相对未开发。一维分析寡核苷酸时，选择离子对试剂通常需要在可接受的色谱性能和质谱性能之间取得平衡。当寡核苷酸被修饰或缀合以包括极疏水基团（例如荧光团）时，由于荧光团对保留的影响，分离机制进一步复杂化。在六氟异丙醇（HFIP）中缓冲的三乙胺（TEA）是最常用的离子配对剂，用于需要质谱分析的分析，但是与具有相同序列的未缀合寡核苷酸相比，染料缀合失败序列相对于主峰的洗脱顺序不是基于长度的。乙酸己基铵（HAA）为基于长度的分离提供了更有效的离子对，但由于离子抑制，MS响应受到损害。在这项研究中，使用2D-LC / MS可以证明染料偶联的寡核苷酸失败序列可以在第一维中使用强离子配对剂从亲本寡核苷酸中分离出来，并使用较弱但与MS兼容的离子混合物进一步鉴定二维中的配对剂，在一维分析中无法获得结果。进一步来说，在第一维和第二维（IP-RP – IP-RP）中均使用离子对反相色谱法进行的心切割配置将n-1杂质从基于第一维的基于长度的分离转移至使用单四极杆检测器进行身份确认的第二维分析。相同的C在第一维和第二维中均使用₁₈柱化学，以利用由于流动相选择而导致的选择性变化。二维分析中的n-1杂质可以在低纳克水平上检测到，与在一维稀释系列中获得的结果相当，接近仪器检测的极限。这项工作在将来适用于使用高分辨率仪器进行更复杂的杂质分析，其中无法使用一维技术评估更广泛的杂质。