Dilute and shoot

In another application, Granelli et al. developed a method for the extraction of a total of 19 antimicrobials, including tetracyclines, sulfonamides, quinolones, P-lactams, and macrolides, from muscle or kidney samples. Extraction was performed with 70% methanol, and the extracts were then diluted five-fold with water before LC-MS injection. The method was suitable only for 

A 2008 paper has described for the first time a dilute and shoot strategy for the simultaneous extraction of wide variety of residues and contaminants (pesticides, myco-toxins, plant toxins, and veterinary drugs) from different foods (meat, milk, honey, and eggs) and feed matrices. Several antimicrobial classes were included (sulfonamides, quinolones, P-lactams, macrolides, ionophores, tetracyclines, and nitroimidazoles) in the analytical method. Sample extraction was performed with water/acetonitrile or acetone/1% formic acid, but instead of dilution of the extracts before analysis by UPLC-MS/MS, small extract volumes (typically 5 til) were injected to minimize matrix effects. Despite the absence of clean-up steps and the inherent complexity of the different sample matrices, adequate recoveries were obtained for the majority of the ana-lyte/matrix combinations (typical values for antimicrobials were in the range of 70-120%). In addition, the use of UPLC allows high-speed analysis, since all analytes eluted within 9 min. 

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Direct injection of plasma or supernatant after protein precipitation on a short column with a high liquid flow rate is a common method for reducing analysis time in the pharmaceutical industry. The direct injection of a sample matrix is also known as the dilute-and-shoot (DAS) approach.62 DAS can be applied to all types of matrices and approaches and is the simplest sample preparation method with matrix dependency. Direct injection can also be approached through the extraction of eluent from PPT, SPE, and LLE onto a normal phase analytical column. The procedure is called hydrophilic interaction liquid chromatography (HILIC)70110111 and it avoids the evaporation and reconstitution steps that may cause loss of samples from heat degradation and absorption. 

Major Matrix Components Showing Potential for Dilute-and-Shoot (DAS) Approach... 

Liquid samples, other than those that are inherently liquid, can arise from the solid sample extraction techniques described above. As mentioned previously, sometimes a simple dilute-and-shoot approach can be utilized, i.e., add solvent to the sample and then inject directly into the instrument. Other times, evaporation of residual liquid can be utilized—then the sample is either directly injected, or if the sample is evaporated to dryness, a new solvent can be added. Often, however, the residual matrix causes interference and the following techniques can be employed for further sample cleanup. 

Sample preparation refers to a family of solid/liquid handling techniques to extract or to enrich analytes from sample matrices into the final analyte solution. While SP techniques are well documented, few references address the specific requirements for drug product preparations, which tend to use the simple dilute and shoot approach. More elaborate SP is often needed for complex sample matrices (e.g., lotions and creams). Many newer SP technologies such as solid-phase extraction... 

This chapter provides the novice and the experienced analyst with an overview of sample preparation techniques focusing on solid dosage forms. It describes the best practices in the dilute and shoot approach, and the tricks of the trade in grinding, mixing, sonication, dilution and filtration of drug products. Selected case studies of sample preparations for assays and impurity testing are used to illustrate the strategies, trade-offs... 

Obviously, some procedures take less time than others. For example, sample concentration by the purge and trap technique that precedes VOC analyses takes only about 20 minutes. It is performed immediately prior to analysis on a multisample automated concentrator combined with an analytical instrument. The shortest of all sample preparation procedures is the waste dilution procedure, commonly known as dilute and shoot, which takes minutes. It consists of diluting a known volume of a concentrated waste sample in a known volume of a compatible solvent, followed by an injection into a gas chromatograph. 

In the early years of LC-MS/MS application in clinical laboratories, chromatographic separation was looked upon as rather unnecessary with tandem mass spectrometers being understood as extremely selective measuring devices. Thus, many LC-MS/MS methods with minimal degree of chromatographic resolution and analyte retention times close to the void time of the chromatographic systems ( dilute and shoot approaches) have been described. However, from the issues discussed so far, the requirements of proper sample preparation and sufficient chromatographic separation prior to MS/MS detection have become evident. 

Interestingly, in the past few years, we have found that straight dilute and shoot methods often succeed where in the past CS was necessary. We attribute this development to the increased sensitivity offered by present-generation mass spectrometers. In other words, it is possible to avoid on-line clean up by simply injecting a more dilute sample or even starting with smaller plasma aliquots. While this strategy is not permissible for all sample matrices, we have achieved consistent success with plasma. 

McCauley-Myers, D.L. Eichhold, T.H. Bailey, R.E. Dobrozsi, D.J. Best, K.J. Hayes II, J.W. Hoke II, S.H. Rapid Bioanalytical Determination of Dextromethorphan in Canine Plasma by Dilute-and-Shoot Preparation Combined with One Minute per Sample LC-MS/MS Analysis to Optimize Formulations for Drug Delivery, J. Pharm. Biomed. Anal. 23, 825-835 (2000). 

FIGURE 21.3 Recovery of stable-labeled standards spiked in urine. Color indicates recovery, according to the scale on the right. Despite the better recovery of many metabolites with some solid-phase extraction (SPE) cartridges, expediency often dictates a dilute-and-shoot approach (see text). OASIS SPE cartridges were obtained from Waters Corp. and used per manufacturer instructions. 

While very low detection limits are definitely needed for analysis of high-purity water samples, this aspect of ion chromatography should not be overly emphasized. Determination of anions at the low ppm (mg L ) level is quite adequate for a great many samples. In fact, it is quite common to dilute a sample before injection in order to bring the analyte concentration into the desired range. This process is often called dilute and shoot Suppressed and nonsuppressed methods are equally valid for many sample types. 

But sometimes more may be needed, either to protect expensive columns or to make the sample detectable. It is possible that a single injection of the wrong sample can destroy a column through contamination or plugging. Or perhaps a sample component must be enhanced to increase the concentration to a detectable level. Sometimes more is needed than just dilute and shoot . This chapter describes the various sample pretreatment methods, why they are used and where they are used. 

In HPLC-MS coupling, careful sample preparation is recommended. The dilute-and-shoot approach is not possible, especially when handling real-world samples such as environmental and biological samples. In these cases sample preparation techniques (see also Chaps. 12.8 and 12.9) such as liquid-liquid extraction, solid-phase extraction [119], or even affinity techniques (see also Chap. 12.11) directly coupled with LC-MS [120] can be useful. 

Sample preparation for LC-MS comprises cleanup of organic solvent extracts using the procedures described earlier, or simple dilution of the extracts and injection without cleanup, a procedure known as dilute and shoot. ... 

One limitation of these three comparative studies is that the sample preparation procedures were different for all the analytical techniques compared, with or without urine hydrolysis, using liquid-liquid extraction (LLE), solid-phase extraction (SPE), or dilute and shoot, which does not actually allow for rigorous comparison of the respective merits of the hyphenated techniques. However, all three showed that LC-MS(/MS) was at least as efficient as the traditional techniques used for GUS/STA in most toxicology laboratories. 

Filtration and injection or protein precipitation with acetonitrile and injection of the supernatant (the so-called dilute and shoot strategy) can provide a direct means to introduce samples into HPLC (41). However, the lack of a concentration step may limit the detection of some of the most potent drugs, while the absence of a purification step may favor matrix effects, hence false negative results. 

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