A dried urine spot test to simultaneously monitor Mo and Ti levels using solid sampling high-resolution continuum source graphite furnace atomic absorption spectrometry

Abstract

Home-based collection protocols for clinical specimens are actively pursued as a means of improving life quality of patients that require frequent controls, such as patients with metallic prosthesis, for whom monitoring the evolution of Mo and Ti in biological fluids may play a decisive role to detect prosthesis mal-functioning. The collection of biological fluids on clinical filter papers provides a simple way to implement these protocols.

This work explores the potential of solid sampling high-resolution continuum source graphite furnace atomic absorption spectrometry for the simultaneous and direct determination of Mo and Ti in urine, after its deposition onto clinical filter paper, giving rise to a dried urine spot. The approach used for depositing the sample was found crucial to develop a quantitative method, since the filter paper acts as a chromatographic support and produces a differential distribution of the target analytes. Furthermore, the high spreading of urine onto a filter paper results in a small amount of urine per surface unit, and thus, ultimately, in lack of sensitivity. In order to circumvent these problems, the use of an alternative approach based on the use of pre-cut 17 × 19 mm filter paper pieces onto which larger amounts of sample (500 μL) can be retained by single deposition was proposed and evaluated. In this way, an approximately 12-fold increase in sensitivity and a more homogeneous distribution of the target analytes were obtained, permitting the development of a quantification strategy based on the use of matrix-matched urine samples of known analyte concentrations, which were subjected to the same procedure as the samples. Accuracy of this method, which provides LODs of 1.5 μg L^− 1 for Mo and 6.5 μg L^− 1 for Ti, was demonstrated after analysis of urine reference materials. Overall, the performance of the method developed is promising, being likely suitable for determination of other analytes in dried urine spots.

Introduction

Ti has no known biological role in humans and it has long been considered as biologically inert and, therefore, it seems ideal for metallic implants, such as dental and orthopedic replacements (joint, hip or spinal implants, to name a few) [1]. Ti quantification in biological fluids could have been considered as anecdotic until a few years ago, as its measurement did not have any clear clinical interest.

On the other hand, Mo is an essential trace element for plants, animals and microorganisms. Up to date, Mo is considered as practically non-toxic to humans, albeit patients with acute Mo poisoning exhibit symptoms similar to those of Cu deficiency or abnormal sulfur metabolism. Therefore, as discussed before for Ti, Mo has not been normally monitored in clinical samples. Furthermore, these elements, and in particular Ti, are not easy to quantify at basal levels by graphite furnace atomic absorption spectrometry (GFAAS) and or by quadrupole-based inductively coupled plasma-mass spectrometry (Q-ICP-MS) [1].

However, the situation has changed as a consequence of the use of metallic implants in joint-replacement surgery, since the concentration of some metals in blood and/or urine has been considered as potential indicator of implant performance. These implants are made with alloys typically containing Cr, Co, Mo and/or Ti. Thus, the monitoring of these analytes in a variety of clinical specimens, such as serum, urine, whole blood, erythrocytes and joint fluid, has become of great interest [2].

A number of investigations have shown that these elements modestly raise its concentration in these matrices after the implant, but then they reach a plateau when the prosthesis is well functioning [3], [4], [5], [6]. Most importantly, it has been demonstrated that a notable increase in the concentration of these metals may occur in mal-functioning prostheses, including examples where it was not possible to visualize this fail (abnormal wear, corrosion or displacement of the implant) by diagnostic imaging techniques, in spite of the pain referred by the patient. These abnormal metal concentrations proved to be evidence of several kinds of unstable prostheses, which had to be replaced surgically [6], [7], [8], [9]. As a consequence of these cases, increasing attention is paid to the monitoring of these metallic elements in clinical samples [10].

On the other hand, there is a growing interest in the deposition of biological fluids, such as blood [11] or urine [12], on filter papers (FP), giving rise to what is generically called dried matrix spots (DMS). This sampling methodology offers significant advantages in terms of logistics for clinical analysis. In the case of urine, this is certainly not a particularly difficult fluid to collect. However, many urinary metabolites have a poor stability, thus requiring restrictive precautions, especially for sample transport and storage. Therefore, the deposition of urine on clinical filter paper (giving rise to dried urine spots, DUS) becomes an attractive alternative due to the ease of preservation [13]. Furthermore, DUS samples can be easily mailed over distances, even overseas, for analysis at referral centers. This aspect is very interesting in various clinical contexts, such as carrying out large-population based studies [14], or monitoring of chronic patients or patients that have undergone a certain type of surgery (e.g., patients with implants). DUS specimens should be easily prepared at home by the own patients, helping to reduce their number of visits to the clinic, and improving their life quality. As a result, any efforts to develop analytical methods based on DUS samples for as many analytes as possible are regarded with interest in the clinical field.

There are several examples in the literature dealing with the analysis of dried urine on clinical filter paper, but the majority are focused on detection of metabolites and drugs [15]. Very little work has been carried out aiming at elemental monitoring, on DUS [16], [17] (using laser ablation–inductively coupled plasma–mass spectrometry, LA–ICP–MS), or in general on DMS [18], [19]. One of the reasons for this limited work may be that, even though the use of this technique looks very appealing for implementing home-based collection methods [20], it presents a further problem for analysis: the transformation of a relatively simple liquid sample (urine) into a solid one (DUS), which typically requires additional work (e.g., digestion or extraction of the analytes of interest from the filter paper prior to analysis [21]). Instead, the use of solid sampling techniques allowing for the direct analysis of DMS represents an attractive alternative, increasing sample throughput and minimizing risks of losses/contamination, although quantification is not always simple owing to matrix-effects [16], [19], [22], [23].

The purpose of this work is to develop a fast method for the direct and simultaneous determination of Mo and Ti in DUS samples prepared with standardized clinical filter paper. As discussed before, such method could be useful for monitoring the evolution of patients with prostheses. For this purpose, we are exploring the use of solid sampling high-resolution continuum source graphite furnace atomic absorption spectrometry (SS HR CS GFAAS). To the best of the author's knowledge, the use of GFAAS has not been evaluated yet for direct analysis of DUS. However, the potential of “classic” line source GFAAS for direct analysis of solid samples [24], [25], including determination of Pb in blood deposited in FP [18], is well-established. Moreover, this potential has been significantly enhanced with the arrival of HR CS GFAAS [26], [27], [28], which offers improved capabilities for the detection and correction of spectral interferences, as well as for the simultaneous monitoring of several elements under certain conditions [29], [30], [31], although developing simultaneous methods, as will be required in this work, is not always straightforward [29]. This aspect, together with the investigation of the distribution of the target elements on the DUS and the development of a suitable calibration approach, will be the main topics investigated in the current work.